TNF-α-stimulated fibroblasts secrete lumican to promote fibrocyte differentiation.

In healing wounds and fibrotic lesions, fibroblasts and monocyte-derived fibroblast-like cells called fibrocytes help to form scar tissue. Although fibrocytes promote collagen production by fibroblasts, little is known about signaling from fibroblasts to fibrocytes. In this report, we show that fibroblasts stimulated with the fibrocyte-secreted inflammatory signal tumor necrosis factor-α secrete the small leucine-rich proteoglycan lumican, and that lumican, but not the related proteoglycan decorin, promotes human fibrocyte differentiation. Lumican competes with the serum fibrocyte differentiation inhibitor serum amyloid P, but dominates over the fibroblast-secreted fibrocyte inhibitor Slit2. Lumican acts directly on monocytes, and unlike other factors that affect fibrocyte differentiation, lumican has no detectable effect on macrophage differentiation or polarization. α2ß1, αMß2, and αXß2 integrins are needed for lumican-induced fibrocyte differentiation. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, lumican is present at low levels throughout the tissue, whereas patients with advanced disease have pronounced lumican expression in the fibrotic lesions. These data may explain why fibrocytes are increased in fibrotic tissues, suggest that the levels of lumican in tissues may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and indicate that modulating lumican signaling may be useful as a therapeutic for fibrosis.

Fibroblasts secrete Slit2 to inhibit fibrocyte differentiation and fibrosis.

Monocytes leave the blood and enter tissues. In healing wounds and fibrotic lesions, some of the monocytes differentiate into fibroblast-like cells called fibrocytes. In healthy tissues, even though monocytes enter the tissue, for unknown reasons, very few monocytes differentiate into fibrocytes. In this report, we show that fibroblasts from healthy human tissues secrete the neuronal guidance protein Slit2 and that Slit2 inhibits human fibrocyte differentiation. In mice, injections of Slit2 inhibit bleomycin-induced lung fibrosis. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, Slit2 has a widespread distribution whereas, in patients with advanced disease, there is less Slit2 in the fibrotic lesions. These data may explain why fibrocytes are rarely observed in healthy tissues, may suggest that the relative levels of Slit2 present in healthy tissue and at sites of fibrosis may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and may indicate that modulating Slit2 signaling may be useful as a therapeutic for fibrosis.

Gadolinium-containing magnetic resonance image contrast agent promotes fibrocyte differentiation.

Gadolinium-containing magnetic resonance imaging (MRI) contrast agents such as Omniscan are associated with nephrogenic systemic fibrosis (NSF). To determine if Omniscan can affect the differentiation of monocytes into fibroblast-like cells called fibrocytes that are found in the fibrotic lesions of NSF, peripheral blood mononuclear cells (PBMCs) from NSF patients, hemodialysis patients without NSF, and healthy, renally sufficient controls were exposed to Omniscan in a standardized in vitro fibrocyte differentiation protocol. When added to PBMCs, the gadolinium-containing MRI contrast agent Omniscan generally had little effect on fibrocyte differentiation. However, 10(-8) to 10(-3) mg/mL Omniscan reduced the ability of the fibrocyte differentiation inhibitor serum amyloid P (SAP) to decrease fibrocyte differentiation in PBMCs from 15 of 17 healthy controls and one of three NSF patients. Omniscan reduced the ability of SAP to decrease fibrocyte differentiation from purified monocytes, indicating that the Omniscan effect does not require the presence of other cells (such as T cells) in the PBMCs. Omniscan also reduced the ability of a different fibrocyte differentiation inhibitor, interleukin-12, to decrease fibrocyte differentiation. These data suggest that Omniscan interferes with the regulatory action of signals that inhibit the differentiation of monocytes to fibrocytes. J. Magn. Reson. Imaging 2009;30:1284-1288. (c) 2009 Wiley-Liss, Inc.

Pivotal Advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation.

CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes, which are associated with and are at least partially responsible for wound healing and fibrosis in multiple organ systems. Signals regulating fibrocyte differentiation are poorly understood. In this study, we find that when added to human PBMCs cultured in serum-free medium, the profibrotic cytokines IL-4 and IL-13 promote fibrocyte differentiation without inducing fibrocyte or fibrocyte precursor proliferation. We also find that the potent, antifibrotic cytokines IFN-gamma and IL-12 inhibit fibrocyte differentiation. In our culture system, IL-1beta, IL-3, IL-6, IL-7, IL-16, GM-CSF, M-CSF, fetal liver tyrosine kinase 3, insulin growth factor 1, vascular endothelial growth factor, and TNF-alpha had no significant effect on fibrocyte differentiation. IL-4, IL-13, and IFN-gamma act directly on monocytes to regulate fibrocyte differentiation, and IL-12 acts indirectly, possibly through CD16-positive NK cells. We previously identified the plasma protein serum amyloid P (SAP) as a potent inhibitor of fibrocyte differentiation. When added together, the fibrocyte-inhibitory activity of SAP dominates the profibrocyte activities of IL-4 and IL-13. The profibrocyte activities of IL-4 and IL-13 and the fibrocyte-inhibitory activities of IFN-gamma and IL-12 counteract each other in a concentration-dependent manner. These results indicate that the complex mix of cytokines and plasma proteins present in inflammatory lesions, wounds, and fibrosis will influence fibrocyte differentiation.

Endothelial progenitor cells display clonal restriction in multiple myeloma.

BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (> or = 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (> or = 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM.

The long pentraxin PTX3 promotes fibrocyte differentiation.

Monocyte-derived, fibroblast-like cells called fibrocytes are associated with fibrotic lesions. The plasma protein serum amyloid P component (SAP; also known as pentraxin-2, PTX2) inhibits fibrocyte differentiation in vitro, and injections of SAP inhibit fibrosis in vivo. SAP is a member of the pentraxin family of proteins that includes C-reactive protein (CRP; PTX1) and pentraxin-3 (PTX3). All three pentraxins are associated with fibrosis, but only SAP and CRP have been studied for their effects on fibrocyte differentiation. We find that compared to SAP and CRP, PTX3 promotes human and murine fibrocyte differentiation. The effect of PTX3 is dependent on FcγRI. In competition studies, the fibrocyte-inhibitory activity of SAP is dominant over PTX3. Binding competition studies indicate that SAP and PTX3 bind human FcγRI at different sites. In murine models of lung fibrosis, PTX3 is present in fibrotic areas, and the PTX3 distribution is associated with collagen deposition. In lung tissue from pulmonary fibrosis patients, PTX3 has a widespread distribution, both in unaffected tissue and in fibrotic lesions, whereas SAP is restricted to areas adjacent to vessels, and absent from fibrotic areas. These data suggest that the relative levels of SAP and PTX3 present at sites of fibrosis may have a significant effect on the ability of monocytes to differentiate into fibrocytes.

Improved serum-free culture conditions for the differentiation of human and murine fibrocytes.

CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes. Fibrocytes are associated with, and are at least partially responsible for, wound healing and fibrosis in multiple organ systems. In a variety of lesions in vivo, monocytes appear to differentiate into fibrocytes within days. However, in vitro culture conditions can take up to two weeks to generate fibrocytes. In this study, we describe enhanced serum-free conditions that support the rapid differentiation of human and murine fibrocytes. We compared the effect on fibrocyte differentiation of different anti-coagulants used when collecting blood, and for culturing cells, the effects of different commercial media formulations, the addition of a variety of supplements, cell density, conditioned medium, and glass and plastic substrates. We found that both heparin and EDTA were suitable anti-coagulants, but that blood treated with citrate-phosphate dextrose led to a reduced number of fibrocytes. Fibrocyte differentiation was enhanced when the serum-free medium was based on either FibroLife or StemPro formulations. We also found that only positively charged or hydrophilic glass and plastic surfaces provide adequate support for fibrocyte differentiation. Finally, the optimal cell density was 2.5 x 10(5)cells/ml (approximately 800 cells per mm(2)). These results indicate that blood collection, substrates, media, and cell density all influence in vitro fibrocyte differentiation.

Circulating endothelial progenitor cells in multiple myeloma: implications and significance.

Angiogenesis governs the progression of multiple myeloma (MM). Circulating endothelial cells (CECs) contribute to angiogenesis and comprise mature ECs and endothelial progenitor cells (EPCs). The present study sought to characterize CECs and their relation to disease activity and therapeutic response in 31 consecutive patients with MM. CECs, identified as CD34(+)/CD146(+)/CD105(+)/CD11b(-) cells, were 6-fold higher in patients compared to controls and correlated positively with serum M protein and beta(2)-microglobulin. Circulating EPCs displayed late colony formation/outgrowth and capillary-like network formation on matrigel; these processes were inhibited after effective thalidomide treatment. Co-expression of vascular endothelial growth factor receptor-2 (KDR) and CD133 characterized EPCs in MM, and KDR mRNA elevations correlated with M protein levels. In vitro exposure of ECs to thalidomide or its derivative CC-5013 inhibited gene expression of the receptors for transforming growth factor-beta and thrombin. Thus, elevated levels of CECs and EPCs covary with disease activity and response to thalidomide, underscoring the angiogenic aspect of MM and suggesting that angioblastlike EPCs are a pathogenic biomarker and a rational treatment target in MM. The results also highlight the anti-angiogenic properties of thalidomide and CC-5013 and further elucidate possible mechanisms of their effectiveness against MM. (Blood. 2005;105:3286-3294).

Differential, tissue-specific, transcriptional regulation of apolipoprotein B secretion by transforming growth factor beta.

Apolipoprotein B (apoB) is required for the assembly and secretion of triglyceride-rich lipoproteins. ApoB synthesis is constitutive, and post-translational mechanisms modulate its secretion. Transforming growth factor beta (TGF-beta) increased apoB secretion in both differentiated and nondifferentiated Caco-2 cells and decreased secretion in HepG2 cells without affecting apolipoprotein A-I secretion. TGF-beta altered apoB secretion by changing steady-state mRNA levels and protein synthesis. Expression of SMAD3 and SMAD4 differentially regulated apoB secretion in these cells. Thus, SMADs mediate dissimilar secretion of apoB in both the cell lines by affecting gene transcription. We identified a 485-bp element, 55 kb upstream of the apob gene that contains a SMAD binding motif. This motif increased the expression of chloramphenicol acetyltransferase in Caco-2 cells treated with TGF-beta or transfected with SMADs. Hence, TGF-beta activates SMADs that bind to the 485-bp intestinal enhancer element in the apob gene and increase its transcription and secretion in Caco-2 cells. This is the first example showing differential transcriptional regulation of the apob gene by cytokines and dissimilar regulation of one gene in two different cell lines by TGF-beta. In this regulation, the presence of cytokine-responsive motif in the tissue-specific enhancer element confers cell-specific response.