New vessel formation in peritumoral area of squamous cell carcinoma of the head and neck.

BACKGROUND: This study was conducted to investigate the dynamic process of new vessel formation, fundamental for tumor growth and metastasis, in head and neck squamous cell carcinoma (HNSCC). METHODS: We used immunohistochemistry, confocal laser-scanning microscopy, and reverse transcriptase-polymerase chain reaction to study endothelial cell and concomitant pericyte development with markers CD133, CD34, VEGFR-2, CD31, vWF, and STRO-1 in tumor and peritumoral tissues of 18 patients with HNSCC. RESULTS: Highly compressed and structurally abnormal vessels with barely any activity of new vessel formation were found in tumor tissue, whereas the adjacent peritumoral tissue vessels showed a normal architecture with tight endothelial cell-pericyte interaction and a high activity of angiogenesis. Endothelial precursor cells expressing CD133/VEGFR-2 could be incorporated into these newly formed vessels, forming cell clusters from which a thin endothelial lining could emanate. CONCLUSIONS: These data show a high activity of new vessel formation in the peritumoral stroma of HNSCC, with endothelial precursor cells being incorporated into these structures.

Endothelial and myogenic differentiation of hematopoietic progenitor cells in inflammatory myopathies.

Incorporation of circulating hematopoietic progenitor cells (HPCs) into damaged skeletal muscle has been proposed as a novel mechanism of tissue repair complementary to satellite cell-dependent regeneration. We studied the occurrence and myoendothelial differentiation of HPCs in muscle of patients with inflammatory myopathies. Muscle biopsies from untreated patients with dermatomyositis, polymyositis, inclusion body myositis, and controls were investigated for the expression of endothelial (CD31, von Willebrand factor, vascular endothelial growth factor receptor 2), hematopoietic (CD34, CD133, CD45), and myogenic (Pax7, MyoD) markers by immunohistochemistry and reverse-transcriptase-polymerase chain reaction. Confocal laser scanning microscopy was used to visualize coexpression of CD34, CD133, von Willebrand factor, or Pax7 on individual cells. Morphometric analysis revealed significantly increased numbers of CD133 cells per square millimeter in polymyositis and inclusion body myositis compared with controls (p < 0.001); this correlated with the density of CD45 infiltrates (p < 0.001). By confocal laser scanning microscopy, we detected several mononuclear cells that coexpressed either CD34/von Willebrand factor or CD133/Pax7 with or without CD34 reactivity, indicating endothelial or myogenic commitment of some HPCs in skeletal muscle. Rarely, CD133/CD34/Pax7 cells seemed to occupy satellite cell niches or to incorporate into preexisting myofibers. Our findings suggest that circulating HPCs colonize skeletal muscle in inflammatory conditions and provide evidence for in situ myoendothelial differentiation of some of these cells.

Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices.

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.

The presence of MOMA-2+ macrophages in the outer B cell zone and protection of the splenic micro-architecture from LPS-induced destruction depend on secreted IgM.

The role secretory IgM has in protecting splenic tissue from LPS-induced damage was assessed in mice incapable of secreting IgM but able to express surface IgM and IgD. Within seconds after LPS challenge, 99% of the (131)I-labeled LPS was found in the liver and the spleen of both sIgM-deficient and wild-type mice. In the spleen FITC-labeled LPS was found on the surface of 2F8(+) scavenger receptor macrophages localized in the outer marginal zone, while none of the labeled LPS could be detected on marginal zone ER-TR9(+) and MOMA-1(+) macrophages. An additional population of macrophages, MOMA-2(+), were capable of producing C3 locally in the T and B cell zone after LPS challenge. Local C3 production was regulated, as no C3 was found in splenic tissue of unchallenged mice. Interestingly, in the absence of circulating and locally produced secretory IgM, MOMA-2(+) macrophages of the T and B cell zone failed to establish an additional ring of C3-producing macrophages in the outer B cell zone close to the marginal zone upon LPS challenge. The consequence was a massive destruction of the microarchitecture of the spleen where marginal zones disorganized, lymphoid follicles and T cell zones disrupted and follicular DC (FDC) networks disappeared.

Endothelial precursor cells in the synovial tissue of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To find evidence for the presence of endothelial precursor cells, which can induce new vessel formation, in the synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Precursor cells in the synovial tissue of 18 RA patients and 15 OA patients were identified by immunohistochemistry, morphometric analysis, and confocal laser scanning microscopy using the following phenotype markers: CD31, CD34, STRO-1, CD133, vascular endothelial growth factor receptor 2 (VEGFR-2), and CXCR4. The presence of CD31, CD34, CD133, VEGFR-2, and CXCR4 messenger RNA in the synovial tissue was determined by reverse transcriptase-polymerase chain reaction, and the message for CXCR4 was quantified by an RNase protection assay. RESULTS: A population of cells that expressed CD34 on their surface but lacked the endothelial cell marker CD31 was found in the synovial tissue of RA and OA patients. CD34+,CD31- cells were detected in close proximity to STRO-1+ and CD133+ cells, forming cell clusters in the sublining area of the synovial membrane. Within these cell clusters, CD34+,CD31- precursor cells were located on the inside surrounded by STRO-1+ cells and with CD133+ cells on the outside. CD34+ precursor cells in the cell layer expressed high levels of the chemokine receptor CXCR4, while VEGFR-2 was expressed on CD34+ and CD133+ cells, and alpha-smooth muscle actin was expressed on STRO-1+ cells. CONCLUSION: The presence of endothelial precursor cells in the synovial tissue of RA and OA patients provides evidence for vasculogenesis induced by precursor cells that arise in situ or from circulating progenitors.