De novo Vessel Formation Through Cross-Talk of Blood-Derived Cells and Mesenchymal Stromal Cells in the Absence of Pre-existing Vascular Structures.

BACKGROUND: The generation of functional blood vessels remains a key challenge for regenerative medicine. Optimized in vitro culture set-ups mimicking the in vivo perivascular niche environment during tissue repair may provide information about the biological function and contribution of progenitor cells to postnatal vasculogenesis, thereby enhancing their therapeutic potential. AIM: We established a fibrin-based xeno-free human 3D in vitro vascular niche model to study the interaction of mesenchymal stromal cells (MSC) with peripheral blood mononuclear cells (PBMC) including circulating progenitor cells in the absence of endothelial cells (EC), and to investigate the contribution of this cross-talk to neo-vessel formation. MATERIALS AND METHODS: Bone marrow-derived MSC were co-cultured with whole PBMC, enriched monocytes (Mo), enriched T cells, and Mo together with T cells, respectively, obtained from leukocyte reduction chambers generated during the process of single-donor platelet apheresis. Cells were embedded in 3D fibrin matrices, using exclusively human-derived culture components without external growth factors. Cytokine secretion was analyzed in supernatants of 3D cultures by cytokine array, vascular endothelial growth factor (VEGF) secretion was quantified by ELISA. Cellular and structural re-arrangements were characterized by immunofluorescence and confocal laser-scanning microscopy of topographically intact 3D fibrin gels. RESULTS: 3D co-cultures of MSC with PBMC, and enriched Mo together with enriched T cells, respectively, generated, within 2 weeks, complex CD31+/CD34+ vascular structures, surrounded by basement membrane collagen type-IV+ cells and matrix, in association with increased VEGF secretion. PBMC contained CD31+CD34+CD45dimCD14- progenitor-type cells, and EC of neo-vessels were PBMC-derived. Vascular structures showed intraluminal CD45+ cells that underwent apoptosis thereby creating a lumen. Cross-talk of MSC with enriched Mo provided a pro-angiogenic paracrine environment. MSC co-cultured with enriched T cells formed "cell-in-cell" structures generated through internalization of T cells by CD31+CD45 dim⁣/ - cells. No vascular structures were detected in co-cultures of MSC with either Mo or T cells. CONCLUSION: Our xeno-free 3D in vitro vascular niche model demonstrates that a complex synergistic network of cellular, extracellular and paracrine cross-talk can contribute to de novo vascular development through self-organization via co-operation of immune cells with blood-derived progenitor cells and MSC, and thereby may open a new perspective for advanced vascular tissue engineering in regenerative medicine.

Comparative Effect of MSC Secretome to MSC Co-culture on Cardiomyocyte Gene Expression Under Hypoxic Conditions in vitro.

INTRODUCTION: Despite major leaps in regenerative medicine, the regeneration of cardiomyocytes after ischemic conditions remains to elucidate. It is crucial to understand hypoxia induced cellular mechanisms to provide advanced treatment options, including the use of stem cell paracrine factors for myocardial regeneration. MATERIALS AND METHODS: In this study, the regenerative potential of hypoxic human cardiomyocytes (group Hyp-CMC) in vitro was evaluated when co-cultured with human bone-marrow derived MSC (group Hyp-CMC-MSC) or stimulated with the secretome of MSC (group Hyp-CMC-SMSC). The secretome of normoxic MSC and CMC, and the hypoxic CMC was analyzed with a cytokine panel. Gene expression changes of HIF-1α, proliferation marker Ki-67 and cytokinesis marker RhoA over different reoxygenation time periods of 4, 8, 24, 48, and 72 h were analyzed in comparison to normoxic CMC and MSC. Further, the proinflammatory cytokine IL-18 protein expression change, metabolic activity and proliferation was assessed in all experimental setups. RESULTS AND CONCLUSION: HIF-1α was persistently overexpressed in Hyp-CMC-SMSC as compared to Hyp-CMC (except at 72 h). Hyp-CMC-MSC showed a weaker HIF-1α expression than Hyp-CMC-SMSC in most tested time points, except after 8 h. The Ki-67 expression showed the strongest upregulation in Hyp-CMC after 24 and 48 h incubation, then returned to baseline level, while a temporary increase in Ki-67 expression in Hyp-CMC-MSC at 4 and 8 h and at 48 h in Hyp-CMC-SMSC could be observed. RhoA was increased in normoxic MSCs and in Hyp-CMC-SMSC over time, but not in Hyp-CMC-MSC. A temporary increase in IL-18 protein expression was detected in Hyp-CMC-SMSC and Hyp-CMC. Our study demonstrates timely dynamic changes in expression of different ischemia and regeneration-related genes of CMCs, depending from the culture condition, with stronger expression of HIF-1α, RhoA and IL-18 if the hypoxic CMC were subjected to the secretome of MSCs.

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System.

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix. Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro, and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model. Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA. Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45+ leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures. Conclusion: Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth.

The secretome of apoptotic human peripheral blood mononuclear cells attenuates secondary damage following spinal cord injury in rats.

After spinal cord injury (SCI), secondary damage caused by oxidative stress, inflammation, and ischemia leads to neurological deterioration. In recent years, therapeutic approaches to trauma have focused on modulating this secondary cascade. There is increasing evidence that the success of cell-based SCI therapy is due mainly to secreted factors rather than to cell implantation per se. This study investigated peripheral blood mononuclear cells as a source of factors for secretome- (MNC-secretome-) based therapy. Specifically, we investigated whether MNC-secretome had therapeutic effects in a rat SCI contusion model and its possible underlying mechanisms. Rats treated with MNC-secretome showed substantially improved functional recovery, attenuated cavity formation, and reduced acute axonal injury compared to control animals. Histological evaluation revealed higher vascular density in the spinal cords of treated animals. Immunohistochemistry showed that MNC-secretome treatment increased the recruitment of CD68(+) cells with concomitant reduction of oxidative stress as reflected by lower expression of inducible nitric oxide synthase. Notably, MNC-secretome showed angiogenic properties ex vivo in aortic rings and spinal cord tissue, and experiments showed that the angiogenic potential of MNC-secretome may be regulated by CXCL-1 upregulation in vivo. Moreover, systemic application of MNC-secretome activated the ERK1/2 pathway in the spinal cord. Taken together, these results indicate that factors in MNC-secretome can mitigate the pathophysiological processes of secondary damage after SCI and improve functional outcomes in rats.

Human blood monocytes support persistence, but not replication of the intracellular pathogen C. pneumoniae.

BACKGROUND: Intracellular pathogens have devised various mechanisms to subvert the host immune response in order to survive and replicate in host cells. Here, we studied the infection of human blood monocytes with the intracellular pathogen C. pneumoniae and the effect on cytokine and chemokine profiles in comparison to stimulation with LPS. RESULTS: Monocytes purified from peripheral blood mononuclear cells by negative depletion were infected with C. pneumoniae. While immunofluorescence confirmed the presence of chlamydial lipopolysaccharide (LPS) in the cytoplasm of infected monocytes, real-time PCR did not provide evidence for replication of the intracellular pathogen. Complementary to PCR, C. pneumoniae infection was confirmed by an oligonucleotide DNA microarray for the detection of intracellular pathogens. Raman microspectroscopy revealed different molecular fingerprints for infected and non-infected monocytes, which were mainly due to changes in lipid and fatty acid content. Stimulation of monocytes with C. pneumoniae or with LPS induced similar profiles of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, but higher levels of IL-1ß, IL-12p40 and IL-12p70 for C. pneumoniae which were statistically significant. C. pneumoniae also induced release of the chemokines MCP-1, MIP-1α and MIP-1ß, and CXCL-8, which correlated with TNF-α secretion. CONCLUSION: Infection of human blood monocytes with intracellular pathogens triggers altered cytokine and chemokine pattern as compared to stimulation with extracellular ligands such as LPS. Complementing conventional methods, an oligonucleotide DNA microarray for the detection of intracellular pathogens as well as Raman microspectroscopy provide useful tools to trace monocyte infection.

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.

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.

Truncated ALK derived from chromosomal translocation t(2;5)(p23;q35) binds to the SH3 domain of p85-PI3K.

The chromosomal translocation t(2;5)(p23;q35) is associated with "Anaplastic large cell lymphomas" (ALCL), a Non Hodgkin Lymphoma occurring in childhood. The fusion of the tyrosine kinase gene-ALK (anaplastic lymphoma kinase) on chromosome 2p23 to the NPM (nucleophosmin/B23) gene on chromosome 5q35 results in a 80 kDa chimeric protein, which activates the "survival" kinase PI3K. However, the binding mechanism between truncated ALK and PI3K is poorly understood. Therefore, we attempted to elucidate the molecular interaction between ALK and the regulatory p85 subunit of PI3K. Here we provide evidence that the truncated ALK homodimer binds to the SH3 domain of p85. This finding may be useful for the development of a new target-specific intervention.

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.