We investigated the effect of interleukin-1 (IL-1) activation of human umbilical vein endothelium (HUVE) on human monocyte transendothelial migration induced by chemotactic factors. Monocyte migration across unactivated endothelium in response to macrophage inflammatory protein-1 alpha (MIP-1 alpha), RANTES, platelet-activating factor (PAF), or monocyte chemoattractant protein-1 (MCP-1) was completely inhibited (90%) by monoclonal antibodies (mAbs; 60.3) to CD18 of the CD11/CD18 complex on the monocyte and partially inhibited (by 75%) in response to C5a. When the HUVE was stimulated with IL-1 alpha (5 h, 0.1 ng/ml), monocyte migration in response to C5a, MIP-1 alpha, RANTES, or PAF was no longer inhibited by mAb to CD18. However, migration was blocked by the combination of mAb to the alpha 4-integrin (CD49d) chain of very late antigen-4 (CD49d/CD29) with the mAb to CD18. In contrast to the above stimuli, activation of the HUVE with IL-1 alpha inhibited the transendothelial migration of monocytes in response to MCP-1. mAbs to the adhesion molecules up-regulated on HUVE by IL-1, i.e., E-selectin (CD62E), intercellular adhesion molecule-1 (CD54) or vascular cell adhesion molecule-1 (CD106), did not reverse the inhibitory effect. Transendothelial migration in response to MCP-1 but not to C5a was inhibited by the treatment of monocytes with culture supernatant from IL-1 alpha-stimulated (but not from unstimulated) HUVE. Such supernatant contained chemotactic activity for monocytes, and a mAb to MCP-1 blocked the migration inhibitory effect of IL-1 activation of the HUVE monolayer, as well as the chemotactic activity in the supernatant from IL-1-stimulated HUVE. The inhibitory effect on migration of IL-1-stimulated HUVE was specific for monocytes because polymorphonuclear leukocyte transendothelial migration in response to IL-8 (a related chemokine) was not inhibited by IL-1 activation of HUVE.(ABSTRACT TRUNCATED AT 250 WORDS)

Controversies and risks continue to be reported about exogenous mesenchymal stem cell-based therapies. In contrast with employing exogenous stem cells, making use of lung resident mesenchymal stem cells (LR-MSCs) could be advantageous. Our study sought to isolate the LR-MSCs and explore their potential to differentiate into alveolar epithelial type II cells (ATII cells). Total lung cells were first precultured, from which the Sca-1(+) CD45(-) CD31(-) population was purified using fluorescence activated cell sorting (FACS). By these methods, it would seem that the Sca-1(+) CD45(-) CD31(-) cells were LR-MSCs. Similar to bone marrow derived mesenchymal stem cells (BM-MSCs), these cells express Sca-1, CD29, CD90, CD44 and CD106, but not CD31 or CD45. They share the same gene expression file with the BM-MSCs and have a similar DNA content during long-term culturing. Furthermore, they could be serially passaged with all these properties being sustained. Above all, LR-MSCs could differentiate into ATII cells when co-cultured with ATII cells in a trans-well system. These findings demonstrated that the Sca-1(+) CD45(-) CD31(-) cells appear to be LR-MSCs that can differentiate into ATII cells. This approach may hold promise for their use in the treatment of lung disease.

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding α4β1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic α4β1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by α4β1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 T-cell survival.

CONCLUSIONS

VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to α4β1 expressing proinflammatory T lymphocytes in CLD.

Bone tissue has a significant potential for healing, which involves a significant the interplay between bone and immune cells. While fracture healing represents a useful model to investigate endochondral bone healing, intramembranous bone healing models are yet to be developed and characterized. In this study, a micro-computed tomography, histomorphometric and molecular (RealTimePCRarray) characterization of post tooth-extraction alveolar bone healing was performed on C57Bl/6 WT mice. After the initial clot dominance (0 h), the development of a provisional immature granulation tissue is evident (7 d), characterized by marked cell proliferation, angiogenesis and inflammatory cells infiltration; associated with peaks of growth factors (BMP-2-4-7,TGFβ1,VEGFa), cytokines (TNFα, IL-10), chemokines & receptors (CXCL12, CCL25, CCR5, CXCR4), matrix (Col1a1-2, ITGA4, VTN, MMP1a) and MSCs (CD105, CD106, OCT4, NANOG, CD34, CD146) markers expression. Granulation tissue is sequentially replaced by more mature connective tissue (14 d), characterized by inflammatory infiltrate reduction along the increased bone formation, marked expression of matrix remodeling enzymes (MMP-2-9), bone formation/maturation (RUNX2, ALP, DMP1, PHEX, SOST) markers, and chemokines & receptors associated with healing (CCL2, CCL17, CCR2). No evidences of cartilage cells or tissue were observed, strengthening the intramembranous nature of bone healing. Bone microarchitecture analysis supports the evolving healing, with total tissue and bone volumes as trabecular number and thickness showing a progressive increase over time. The extraction socket healing process is considered complete (21 d) when the dental socket is filled by trabeculae bone with well-defined medullary canals; it being the expression of mature bone markers prevalent at this period. Our data confirms the intramembranous bone healing nature of the model used, revealing parallels between the gene expression profile and the histomorphometric events and the potential participation of MCSs and immune cells in the healing process, supporting the forthcoming application of the model for the better understanding of the bone healing process.

Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been reported. Utilizing standard in vitro and in vivo assays we performed a side-by-side comparison of MEF and BMSC to determine their ability to differentiate into mesoderm-type cells. BMSC were isolated from 8-10 weeks old mouse bone marrow by plastic adherence. MEF were established by trypsin/EDTA digestion from E13.5 embryos after removing heads and viscera, followed by plastic adherence. Compared to BMSC, MEF exhibited telomerase activity and improved cell proliferation as assessed by q-PCR based TRAP assay and cell number quantification, respectively. FACS analysis revealed that MEF exhibited surface markers characteristic of the BMSC: Sca-1(+), CD73(+), CD105(+), CD29(+), CD44(+), CD106(+), CD11b(-), and CD45(-). In contrast to BMSC, ex vivo osteoblast (OB) differentiation of MEF exhibited a less mature osteoblastic phenotype (less alkaline phosphatase, collagen type I and osteocalcin) as assessed by real-time PCR analysis. Compared to BMSC, MEF exhibited a more enhanced differentiation into adipocyte and chondrocyte lineages. Interestingly, both MEF and BMSC formed the same amount of heterotopic bone and bone marrow elements upon in vivo subcutaneous implantation with hydroxyapatite/tricalcium phosphate, in immune deficient mice. In conclusion, MEF contain a population of stem cells that behave in ex vivo and in vivo assays, similar but not identical, to BMSC. Due to their enhanced cell growth, they may represent a good alternative for BMSC in studying molecular mechanisms of stem cell commitment and differentiation to osteoblasts, adipocytes and chondrocytes.

BACKGROUND

The aim of the study was to characterize synovial cells from OA synovium with low-grade and moderate-grade synovitis and to define the role of synovial macrophages in cell culture.

METHODS

Synovial tissue explants were analyzed for the expression of typical markers of synovial fibroblasts (SF), synovial macrophages (SM) and endothelial cells. Synovial cells at passage 1 (p.1) and 5 (p.5) were analyzed for different phenotypical markers by flow cytometric analysis, inflammatory factors by multiplex immunoassay, anabolic and degradative factors by qRT-PCR. P.1 and p.5 synovial cells as different cell models were co-cultured with adipose stem cells (ASC) to define SM effects.

RESULTS

Synovial tissue showed a higher percentage of CD68 marker in moderate compared with low-grade synovitis. Isolated synovial cells at p.1 were positive to typical markers of SM (CD14, CD16, CD68, CD80 and CD163) and SF (CD55, CD73, CD90, CD105, CD106), whereas p.5 synovial cells were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and some anabolic (IL10) factors than those of p.5. Moreover, p.1 synovial cells also expressed a higher amount of some degradative factors (MMP13, S100A8, S100A9) than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells differently induced or down-modulated some of the inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and degradative factors (ADAMTS5, MMP13, S100A8, S100A9).

CONCLUSIONS

We found that p.1 (mix of SM and SF) and p.5 (only SF) synovial cells represent two cell models that effectively reproduce the low- or moderate-grade synovitis environment. The presence of SM in culture specifically induces the modulation of the different factors analyzed, confirming that SM are key effector cells.

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.

Adipose tissue contains some populations, adipose-derived stem cells (ADSCs) which can differentiate into adipogenic, chondrogenic, osteogenic, myogenic, and endothelial cells. Furthermore, adipose tissue can be easily obtained in large quantities through a simple liposuction. ADSCs are thought to be an alternate source of autologous adult stem cells for cell-based therapy. However, it is time-consuming and inefficient to harvest ADSCs by using a traditional collagenase-digestion method. To meet the demand of large quantities of ADSCs in the basic and applied research of regenerative medicine, we developed a rapid and efficient method for isolation and culture of primary ADSCs. The results indicated that the ADSCs obtained with our method possessed strong abilities of proliferation and colony formation in vitro, and could keep low level of cell senescence with stable population doubling during long-term culture in vitro. Furthermore, these harvested ADSCs were capable to differentiate into osteogenic and adipogenic lineages in the specific induction medium. In addition, the results of flow cytometry analysis indicated that these ADSCs could positively express multiple CD markers, such as CD44, CD105, CD29, CD90, and CD13, and hardly expressed CD31, CD34, CD45, and CD106, which was homologous to the mesenchymal stem cells. Therefore, the ADSCs isolated with our method are consistent with previously reported characteristics of the ADSCs. This new method that we established in this study is an efficient tool to isolate and culture the stem cells from adipose tissue.

BACKGROUND

The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

RESULTS

By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7-14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

CONCLUSIONS

Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

OBJECTIVE

To evaluate whether microbubbles targeted to vascular cell adhesion molecule-1 (VCAM-1) (CD106) coupled with a magnetic guidance system could improve the efficacy of contrast-enhanced molecular ultrasonography (US) of atherosclerosis in the aorta.

METHODS

The animal research committee at Southern Medical University approved all experiments. Adherence of magnetic VCAM-1-targeted microbubbles, control inactive magnetic microbubbles, and nonmagnetic VCAM-1-targeted microbubbles to VCAM-1-Fc was determined in vitro by using a flow chamber at variable shear stress (1-24 dyne/cm(2)) under magnetic field guidance. Attachment of microbubbles under magnetic field guidance was determined in vivo with fluorescent microscopy and contrast-enhanced US of the abdominal aorta in wild-type (C57BL/6) or apolipoprotein E (APOE)-deficient mice on a regular or hypercholesterolemic diet. General factorial analysis of variance was used to compare the targeted effect of the microbubbles among different animal groups to identify significant differences.

RESULTS

Attachment was noted for magnetic and nonmagnetic microbubbles but not for inactive magnetic microbubbles; firm attachment at high shear stress (16-20 dyne/cm(2)) was achieved only with magnetic microbubbles. Fluorescence intensity and video intensity were significantly higher in magnetic microbubbles with magnetic field guidance than in inactive magnetic microbubbles and nonmagnetic microbubbles (P < .05). Video intensity from retained magnetic microbubbles in APOE-deficient mice was significantly greater than that in wild-type mice (mean video intensity for APOE-deficient mice: 28.25 [interquartile range, or IQR, 26.55-29.20] with a hypercholesterolemic diet and 16.10 [IQR, 14.15-18.75] with a regular diet; mean video intensity for wild-type mice: 9.55 [IQR, 8.85-10.5] with a hypercholesterolemic diet and 2.90 [IQR, 1.25-3.85] with a regular diet; P < .001).

CONCLUSIONS

Use of a magnetic targeted microbubble system results in greater attachment to endothelial VCAM-1 in atherosclerotic aortas in conditions of high shear stress and improved detection of early inflammatory changes of atherosclerosis.

Although fumaric acid esters (FAE) have a decade-long firm place in the therapeutic armamentarium for psoriasis, their pleiotropic mode of action is not yet fully understood. While most previous studies have focused on the effects of FAE on leucocytes, we have addressed their activity on macro- and microvascular endothelial cells. As detected both on mRNA and protein levels, dimethylfumarate effected a profound reduction of TNFα-induced expression of E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106) on two different endothelial cell populations in a concentration-dependent manner. This reduction of several endothelial adhesion molecules was accompanied by a dramatic diminution of both rolling and firm adhesive interactions between endothelial cells and lymphocytes in a dynamic flow chamber system. Dimethylfumarate, at a concentration of 50 μm, reduced lymphocyte rolling on endothelial cells by 85.9% (P<0.001 compared to untreated controls), and it diminished the number of adherent cells by 88% (P<0.001). In contrast, monomethylfumarate (MMF) influenced neither surface expression of adhesion molecules nor interactions between endothelial cells and lymphocytes. These observations demonstrate that endothelial cells, in addition to the known effects on leucocytes, undergo profound functional changes in response to dimethylfumarate. These changes are accompanied by severely impaired dynamic interactions with lymphocytes, which constitute the critical initial step of leucocyte recruitment to inflamed tissues in psoriasis and other TNF-related inflammatory disorders.

BACKGROUND

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) may play an important role in inflammation, because it can hydrolyze the GPI anchors of several inflammatory membrane proteins (eg, CD106, CD55, and CD59) and its hydrolytic products upregulate macrophage cytokine expression (eg, interleukin-1 and tumor necrosis factor-alpha). Because of its potential regulatory role in inflammatory reactions, we hypothesized that GPI-PLD might be expressed in atherosclerosis.

RESULTS

Immunohistochemistry using human GPI-PLD-specific rabbit polyclonal antiserum was performed on a total of 83 nonatherosclerotic and atherosclerotic human coronary arteries from 23 patients. Macrophages, smooth muscle cells, apoA-I, and oxidation epitopes also were identified immunohistochemically. Cell-associated GPI-PLD was detected in 95% of atherosclerotic segments, primarily on a subset of macrophages. Extracellular GPI-PLD was present in only 30% of atherosclerotic segments and localized to regions with extracellular apoA-I. In contrast, GPI-PLD was not detected in nonatherosclerotic segments. Expression of GPI-PLD mRNA by human macrophages was confirmed in vitro by reverse transcription/polymerase chain reaction. Further studies demonstrated that GPI-PLD-positive plaque macrophages contained oxidation epitopes, suggesting a link between oxidant stress and GPI-PLD expression. This possibility was supported by studies in which exposure of a macrophage cell line to H2O2 led to a 50+/-3% increase in steady-state GPI-PLD mRNA levels.

CONCLUSIONS

Collectively, these results suggest that oxidative processes may regulate GPI-PLD expression and suggest a role for GPI-PLD in inflammation and in the pathogenesis of atherosclerosis.

The adherence of monocytes to activated endothelium is an early event in atherogenesis. Because antioxidants have been considered to be of antiatherosclerotic potential, we investigated the effects of alpha-tocopherol (TCP) and its acetate and succinate esters on monocyte adhesion to cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Endothelial cells were treated with TCP, alpha-tocopherol acetate (TCP acetate), or alpha-tocopheryl succinate (TCP succinate) before stimulation with tumor necrosis factor-alpha (TNF-alpha; 10 U/ml, 6 h) or interleukin-1 beta (IL-1 beta; 10 U/ml, 6 h). Cytokine-stimulated cell surface expression of vascular cell adhesion molecule-1 (VCAM-1, CD106) and E-selectin (ELAM-1, CD62E), but not of intercellular adhesion molecule-1 (ICAM-1, CD54), was time- and dose-dependently inhibited by TCP succinate but not by TCP or TCP acetate. TCP succinate (200 microM, 24 h) reduced TNF-induced VCAM-1 and E-selectin expression from a specific mean fluorescence intensity of 151 +/- 28 to 12 +/- 4 channels and from 225 +/- 38 to 79 +/- 21 channels, respectively. Succinate alone had no effect. Decreased adhesion molecule expression was associated with a reduction of monocytic cell adhesion. TCP succinate (20 microM, 72 h), but not TCP (200 microM, 72 h), reduced U-937 cell adhesion to TNF-alpha-stimulated (10 U/ml, 6 h) HUVEC by 30% (P < 0.025) and to IL-1 beta-stimulated HUVEC by 56% (P < 0.010). Electrophoretic mobility-shift assays of HUVEC nuclear proteins revealed a decrease in TNF-alpha-stimulated nuclear factor-kappa B (NF-kappa B) activation after pretreatment of HUVEC with TCP succinate but not with TCP, TCP acetate, or succinate alone. In conclusion, we demonstrate that the vitamin E derivative TCP succinate prevents monocytic cell adhesion to cytokine-stimulated endothelial cells by inhibiting the activation of NF-kappa B, further emphasizing the antiatherosclerotic potential of lipid soluble antioxidants.

Type 1 diabetes can be cured by transplantation of isolated pancreatic islets. Because of the shortage of human donor tissue, adult porcine islets (APIs) constitute a possible alternative tissue source. Upon intraportal injection, islets are subjected to an instant blood-mediated inflammatory reaction (IBMIR) leading to blood clotting, leukocyte islet-infiltration, islet damage and insulin release. Xenogeneic islets surviving IBMIR are rejected in a cellular process involving CD4(+) T lymphocytes and macrophages. We have investigated whether APIs themselves produce and secrete chemokines and/or inflammatory cytokines that may contribute to IBMIR and/or cell-mediated rejection. APIs, cultured for 1, 4, 8 and 11 days post-isolation, expressed mRNA for monocyte chemoattractant protein-1 (MCP-1), IL-1beta and TNF-alpha. API culture supernatants induced migration of human monocytes, which was significantly blocked by an anti-human MCP-1 antibody (Ab). Immunohistochemistry revealed MCP-1 in the cytoplasm of alpha- and beta-cells in isolated islets and in islets in situ. However, APIs or their supernatants were not able to activate human aortic endothelial cells (HAECs) in vitro, and neither IL-1beta nor TNF-alpha were detected by enzyme-linked immunosorbent assay (ELISA) in API culture supernatants. Both recombinant porcine IL-1beta and TNF-alpha were able to activate human endothelial cells (ECs) inducing CD62E and CD106 expression as analyzed by flow cytometry. In conclusion, MCP-1 secreted by APIs may contribute to both IBMIR and rejection by attracting monocytes into the islet; monocytes which upon transformation into macrophages will potentiate antigen presentation and execute islet rejection.

A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cells had normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the same conditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stained negatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive after osteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining. After osteogenic induction, ossification nodules of goat ADSCs were larger than in rats, with dense ALP staining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor (PPARγ2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. More cartilage lacunae with better morphology were seen following differentiation of goat ADSC's using the hang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop induced cultures were better than for three-dimensional cultures.

Interleukin-10 (IL-10) is an anti-inflammatory helper T cell type 2 (Th2) cytokine that modulates Th1-type cytokine production. Graft arterial disease (GAD) is a vascular obliterative process mediated via the Th1 cytokine interferon-gamma (IFN-gamma); allografts in IFN-gamma-deficient animals do not develop GAD. We investigated the effect of IL-10 and anti-IL-10 on GAD in murine heart transplants and whether anti-IL-10 reestablishes GAD in IFN-gamma-deficient hosts. Major histocompatibility complex class II-mismatched hearts were transplanted for 8 weeks into wild-type or IFN-gamma-deficient mice. In one set of experiments, wild-type hosts received daily administration of phosphate-buffered saline (PBS) or increasing IL-10; in a subsequent set of experiments, wild-type hosts received weekly PBS, rat IgG, or anti-IL-10 monoclonal antibody; IFN-gamma-deficient recipients received weekly PBS or anti-IL-10 monoclonal antibody. Explanted allografts were assessed for parenchymal rejection and GAD, cytokine profiles, and adhesion/costimulatory-molecule expression. Exogenous IL-10 resulted in increased Th2-like cytokine production; nevertheless, it exacerbated parenchymal rejection and GAD and increased CD8(+) infiltration. Anti-IL-10 did not significantly affect the extent of rejection or GAD, cytokine profiles, or immunohistology of the allografts in wild-type hosts. Adhesion molecule (CD54 and CD106) expression was not diminished by IL-10 treatment, and costimulatory-molecule (CD80 and CD86) expression was augmented by administration of exogenous IL-10. Allografts in IFN-gamma-deficient recipients showed mild rejection and no GAD, regardless of anti-IL-10 treatment. IL-10 in vivo thus has markedly different effects than predicted from in vitro experience. Although allografts develop Th2-like cytokine profiles treatment with IL-10 causes exacerbated rejection and GAD.

BACKGROUND

The extracellular matrix is an essential microenvironment for cell survival activity. The adipose tissue extract microparticle scaffolds from human adipose tissue and small intestine submucosa microparticle scaffolds from porcine jejunum were prepared. Their effects on the adipogenic capabilities of human adipose-derived stem cells were compared in vivo.

METHODS

A combination of physical and chemical methods was used to decellularize human fat and porcine jejunum. Expression of CD molecules on the adipose-derived stem cell surface was determined by flow cytometry. The stem cells were then cultured with the scaffold materials in vitro. The cell-scaffold complexes were implanted subcutaneously into nude mice, and samples were collected 4 and 8 weeks later. The adipogenic differentiation capabilities of adipose-derived stem cells were studied by histologic methods and real-time polymerase chain reaction.

RESULTS

The authors observed high expression of CD90 and CD44; no expression of CD34, CD45, CD31, or CD106; and weak positive expression of CD49d on the extracted cells, which indicates that the cells were adipose-derived stem cells. The main constituent of the decellularized adipose tissue extract and small intestine submucosa microparticles was collagenous fiber, and the cells proliferated faster on the adipose tissue extract than on small intestine submucosa. Formation of adipocytes in the adipose tissue extract group was closer to that of normal human fat tissue compared with that of the small intestine submucosa group.

CONCLUSIONS

Extracellular matrix microparticle scaffolds could promote proliferation, adhesion, and adipogenic differentiation of adipose-derived stem cells. The role of the adipose tissue extract microparticle scaffold in promoting adipogenesis was stronger and more suitable as a vector in fatty tissue engineering.

Renal resident mesenchymal stem cells (MSCs) are important regulators of kidney homeostasis, repair or regeneration. However, natural distribution and the starting population properties of these cells remain elusive because of the lack of specific markers. Here, we identified post-natal kidney derived Nestin(+) cells that fulfilled all of the criteria as a mesenchymal stem cell. These isolated Nestin(+) cells expressed the typical cell-surface marker of MSC, including Sca-1, CD44, CD106, NG2 and PDGFR-α. They were capable of self-renewal, possessed high clonogenic potential and extensive proliferation for more than 30 passages. Under appropriate differentiation conditions, these cells could differentiate into adipocytes, osteocytes, chondrocytes and podocytes. After intravenous injection into acute kidney injury mice, Nestin(+) cells contributed to functional improvement by significantly decreasing the peak level of serum creatinine and BUN, and reducing the damaged cell apoptosis. Furthermore, conditioned medium from Nestin(+) cells could protect against ischemic acute renal failure partially through paracrine factor VEGF. Taken together, our findings indicate that renal resident Nestin(+) MSCs can be derived, propagated, differentiated, and repair the acute kidney injury, which may shed new light on understanding MSCs biology and developing cell replacement therapies for kidney disease.

BACKGROUND

Umbilical cord blood (UCB) has been widely used for hematopoietic stem cell transplantation. The UCB-derived stem cells (UCBSCs) have been proposed as an alternative to bone marrow (BM)-derived mesenchymal stem cells (MSCs) for cardiac cell-based therapy. Herein we studied whether UCBSCs spontaneously exhibit cardiac-specific markers in vitro.

METHODS

Human UCBSCs were isolated, expanded, and phenotyped by flow cytometry, quantitative RT-PCR, and immunofluorescence. Cell pluripotency and proliferation were also assessed by adipogenic and osteogenic media and in growth assays.

RESULTS

Among 25 analyzed UCB, 16% of cases afforded primary culture satisfactory generation of UCBSCs. Duplication time (Td) of cultures was 2.16 +/- 0.06 days. The cells were strongly positive for CD105 (18.5 +/- 0.14), CD44 (27 +/- 2.8), CD166 (13 +/- 9), CD29 (59 +/- 9.4), CD90 (60 +/- 11) and consistently negative for CD117 (1.2 +/- 0.1), CD106 (1.1 +/- 0), CD34 (1.2 +/- 0.2), CD14 (1 +/- 0), and CD45 (1 +/- 0), consistent with a mesenchymal lineage. Adipogenesis and osteogenesis of cells resulted in low accumulation of intracellular lipid droplets and high deposition of calcium. The UCBSCs showed gene transcripts for alpha-actinin, connexin (Cx)-43, SERCA-2, and stromal cell-derived factor (SDF)-1alpha. At the protein level, the cells abundantly expressed alpha-actinin, Cx-43, SERCA-2 and SDF-1alpha. In contrast, these cells did not express the cardiac transcription factors GATA-4, Tbx5, and Nkx2.5, nor the sarcomeric proteins beta-myosin heavy chain (beta-MyHC) or cardiac troponin I (cTnI).

CONCLUSIONS

Human UCBSCs may represent an alternative source of stem cells for myocardial-cell replacement. These cells can be highly expanded. They spontaneously express proteins of paramount importance for cardiovascular regeneration, such as Cx-43, SERCA-2, and SDF-1alpha.

Monocytes migrate through vascular endothelium, and then in connective tissue. As a model of this process, we investigated adhesion molecules involved in monocyte migration through HUVEC and a barrier of human synovial fibroblasts (HSF). Minimal spontaneous monocyte migration (6-7%) occurred through either cell barrier, but this increased markedly (27-35% of added monocytes) when a C5a chemotactic gradient was present. Migration across unstimulated HUVEC was partially inhibited (40%) by mAb to CD18 (beta2 integrin) and completely blocked by anti-CD18 plus anti-alpha4 (CD49d; very late Ag-4 (VLA-4)) mAbs. In contrast, migration across HSF induced by C5a or monocyte chemoattractant protein-1 was not inhibited by mAb to CD18 and was only partially inhibited (33%) in combination with anti-alpha4 mAb. The CD18- and VLA-4-independent migration across HSF was completely inhibited by mAb to alpha5 of VLA-5. The inhibitory effect of mAbs to VLA-4 and VLA-5 was on the monocyte and required blockade of CD11/CD18 to be observed. In contrast to HSF, no role for VLA-5 in monocyte transendothelial migration was detected. Both HSF and IL-1-stimulated HUVEC expressed vascular cell adhesion molecule-1 (VCAM-1). However, VLA-4-mediated monocyte migration across HSF was only partially dependent on VCAM-1, in contrast to transendothelial migration, which was completely blocked by anti-VCAM-1 mAbs. In conclusion, unlike transendothelial migration, for which VLA-4 is the alternative mechanism to CD11/CD18 on monocytes, both VLA-4 and VLA-5 can mediate monocyte migration through fibroblast barriers. In addition to VCAM-1, other ligand(s) on HSF are also involved in the VLA-4-mediated migration.

Human periodontal ligament stem cells (PDLSCs) are a unique population of mesenchymal stem cells (MSCs) which demonstrate the capacity to generate cementum- and periodontal ligament-like structures in vivo. As such, PDLSCs represent a promising cell-based therapy in reconstructive dentistry for the treatment of periodontal disease. The present chapter describes two methods for isolating PDLSCs from human PDL tissue including traditional plastic adherence and immunomagnetic selection based on the expression of MSC-associated surface markers STRO-1 antigen, CD146 (MUC-18), CD29 (integrin beta-1), CD44, and CD106 (VCAM-1). Although no single antibody demonstrates specificity for MSCs, isolation based on the expression of individual markers results in homogeneous preparations of PDLSCs. Methods to further characterize the immunophenotype and multipotent capacity of PDLSCs to differentiate into adipocytes, osteoblast- and cementoblast-like cells in vitro, and cementum- and periodontal ligament-like tissues in vivo are also described.

OBJECTIVE

Establish a reproducible method for the isolation and cultivation of murine pulmonary microvascular endothelium. To this end, we exploited the localized pattern of microvascular endothelial activation induced in vivo by inflammatory stimuli to isolate a subpopulation of endothelium for in vitro study.

METHODS

Immunohistochemical analyses of the pulmonary vasculature of mice treated systemically with gram-negative bacterial endotoxin (LPS) demonstrated selective expression of VCAM-1 (CD106) in the endothelial lining of small collecting veins, venules, septal capillaries, and, infrequently, small arteries, which was not observed in control mice. Single cell suspensions prepared by enzymatic dissociation of peripheral lobular tissues dissected from the lungs of LPS-stimulated mice were incubated with a phycoerythrin-conjugated antimouse VCAM-1 monoclonal antibody (MK 1.91). Cells expressing this antigen were isolated by sterile fluorescence-activated cell sorting (FACS). Positive cell populations were collected and cultured for 1-2 weeks. When confluent, these primary cultures were further FACS enriched for endothelium, positively selecting for cells incorporating a fluorescent derivative of acetylated low density lipoprotein (Di-I-Ac-LDL).

RESULTS

The resulting population of cells (mouse lung endothelial cells, MLEC) were uniformly positive for the endothelial markers von Willebrand factor, thrombomodulin, and Dil-Ac-LDL uptake. MLEC readily formed tube-like structures when cultured on Matrigel and spontaneously demonstrated a sprouting phenotype on fibronectin or collagen matrices. MLEC retained responsiveness to cytokines (IL-1 alpha, IL-1 beta, TNF alpha, IFN gamma) up to at least eight passages from primary culture and demonstrated upregulation of E-selectin (CD62E) and P-selectin (CD62P) mRNA as early as 2 hr after LPS stimulation. Characteristic temporal expression patterns of cell surface E-selectin (maximal at 4 hr and declining toward baseline by 24 hr), VCAM-1 (maximal at 6-8 hr and remaining elevated for 24-48 hr), and ICAM-1 (maximal at 6-8 hr and maintained at 24 hr) were observed when cultured MLEC were treated with recombinant murine TNF alpha or recombinant human (rh) IL-1 alpha or rhIL-1 beta. The rolling, adhesion, and transmigration of human polymorphonuclear leukocytes was markedly increased on cytokine-activated MLEC monolayers under defined flow conditions.

CONCLUSIONS

The strategy of activation-dependent isolation allows for the reproducible selection of a specific subset of microvascular endothelial cells for in vitro study. This experimental approach should further facilitate study of the functional heterogeneity of endothelium and its pathophysiologic dysfunction.

OBJECTIVE

The aim of the present study was to investigate the effect of a mixture of proteolytic enzymes (comprising trypsin, chymotrypsin and papain) on the metastatic model of syngeneic melanoma B16.

METHODS

140 C57B16 mice were divided into two control and two "treated" groups. Control groups received saline rectally, twice a day starting 24 h after intracutaneous transplantation (C1) or from the time point of the primary B16 melanoma extirpation (C2), respectively. "Treated" groups were rectally administered a mixture of 0.2 mg trypsin, 0.5 mg papain, and 0.2 mg chymotrypsin twice daily starting 24 h after transplantation (E1) or after extirpation of the tumor (E2), respectively. Survival of mice and B16 melanoma generalization were observed for a period of 100 days. Immunological evaluation of B16 melanoma cells in the ascites was accomplished. CD44, CD54 and CD106 cells were measured by flow cytometry.

RESULTS

Administration of proteolytic enzymes to mice inhibited the growth of primary tumors, and tumor recurrences were less numerous. Importantly, metastasis was considerably curtailed both in the vicinity of the primary tumor and at distant locales. These findings correlated with a decreased expression of CD44 and CD54 molecules in tumors exposed to proteolytic enzymes in vivo.

CONCLUSIONS

Our data suggest that serine and cysteine proteinases suppress B16 melanoma, and restrict its metastatic dissemination in C57B16 mice.

BACKGROUND

Chronic lymphocytic leukemia B cells display prolonged survival in vivo, but when cultured in vitro rapidly undergo spontaneous apoptosis. We hypothesize that interactions with endothelial cells in infiltrated tissues and during recirculation may have a pathogenic role in chronic lymphocytic leukemia.

METHODS

We evaluated apoptosis of leukemic cells after co-culture on a monolayer of human umbilical vein endothelial cells with addition of fludarabine and antibodies that block adhesion. Then, we compared microarray-based gene expression profiles between leukemic cells at baseline and after co-culture.

RESULTS

We found that the endothelial layer protected leukemic cells from apoptosis inducing a 2-fold mean decrement in apoptotic cells after 2 days of co-culture. Moreover, the endothelial layer decreased the sensitivity of chronic lymphocytic leukemia B cells to fludarabine-induced apoptosis. Physical contact with endothelium mediated by both β(1)- and β(2)- integrins is essential for the survival advantage of leukemic cells. In particular, blocking CD106 on endothelial cells or CD18 on leukemic B cells led to the almost complete abrogation of the survival advantage (>70% inhibition of viability). However, a reduction of apoptosis was also measured in leukemic cells cultured in conditioned medium collected after 2 days of co-culture, implying that survival is partially mediated by soluble factors. Overall, the contact with endothelial cells modulated 1,944 genes in chronic lymphocytic leukemia B cells, establishing a peculiar gene expression profile: up-regulation of angiogenesis-related genes, an increase of genes involved in TGFβ and Wnt signaling pathways, secretion of cytokines recruiting stromal cells and macrophages and up-regulation of anti-apoptotic molecules such as Bcl2 and Survivin.

CONCLUSIONS

Our study supports the notion that endothelial cells are major players in the chronic lymphocytic leukemia microenvironment. Adhesion to endothelium strongly supports survival, protects from drug-induced apoptosis and extensively modifies the gene expression profile of leukemic cells.