Resistin-like molecule ß acts as a mitogenic factor in hypoxic pulmonary hypertension via the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK signaling pathways.

BACKGROUND: Pulmonary arterial smooth muscle cell (PASMC) proliferation plays a crucial role in hypoxia-induced pulmonary hypertension (HPH). Previous studies have found that resistin-like molecule ß (RELM-ß) is upregulated de novo in response to hypoxia in cultured human PASMCs (hPASMCs). RELM-ß has been reported to promote hPASMC proliferation and is involved in pulmonary vascular remodeling in patients with PAH. However, the expression pattern, effects, and mechanisms of action of RELM-ß in HPH remain unclear. METHODS: We assessed the expression pattern, mitogenetic effect, and mechanism of action of RELM-ß in a rat HPH model and in hPASMCs. RESULTS: Overexpression of RELM-ß caused hemodynamic changes in a rat model of HPH similar to those induced by chronic hypoxia, including increased mean right ventricular systolic pressure (mRVSP), right ventricular hypertrophy index (RVHI) and thickening of small pulmonary arterioles. Knockdown of RELM-ß partially blocked the increases in mRVSP, RVHI, and vascular remodeling induced by hypoxia. The phosphorylation levels of the PI3K, Akt, mTOR, PKC, and MAPK proteins were significantly up- or downregulated by RELM-ß gene overexpression or silencing, respectively. Recombinant RELM-ß protein increased the intracellular Ca2+ concentration in primary cultured hPASMCs and promoted hPASMC proliferation. The mitogenic effects of RELM-ß on hPASMCs and the phosphorylation of PI3K, Akt, mTOR, PKC, and MAPK were suppressed by a Ca2+ inhibitor. CONCLUSIONS: Our findings suggest that RELM-ß acts as a cytokine-like growth factor in the development of HPH and that the effects of RELM-ß are likely to be mediated by the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK pathways.

Exogenous bFGF promotes articular cartilage repair via up-regulation of multiple growth factors.

OBJECTIVE: To investigate the roles of exogenous basic fibroblast growth factor (bFGF) on the repair of full-thickness articular cartilage defects in rabbits. DESIGN: In the present study, a double-layered collagen membrane sandwiched with bFGF-loaded-nanoparticles between a dense layer and a loose layer was implanted into full-thickness articular cartilage defects in rabbits. By grafting the membrane in a different direction, the dense layer or the loose layer facing the surface of the subchondral bone, the effects of the released bFGF on the defects and the profiles of nine growth factors (GFs) in synovial fluid (SF) were investigated using histological methods and antibody arrays, respectively. RESULTS: In the group with the loose layer facing the surface of the subchondral bone, fast release of bFGF was observed, and early high levels of endogenous transforming growth factor-ß2 (TGF-ß2), vascular endothelial growth factor (VEGF), bFGF, bone morphogenetic protein 2 (BMP-2), BMP-3, and BMP-4 in SF were detected by antibody arrays, especially on day 3. Chondrocyte-like cells were also observed in this group at an early stage. As a result, this group showed better levels of repair, as compared to the other groups in which low GF levels were detected at an early stage, and chondrocyte-like cells appeared much later. CONCLUSIONS: Our study suggests that exogenous bFGF promotes articular cartilage repair by up-regulating the levels of multiple GFs, but administration at an early stage is required.

An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination.

We describe a strategy for generating an allelic series of mutations at a given locus that requires the production of only one targetted mouse line. The 'allelogenic' mouse line we produced carries a hypomorphic allele of Fgf8, which can be converted to a null allele by mating to cre transgenic animals. The hypomorphic allele can also be reverted to wild-type by mating the allelogenic mice to flp transgenic animals, thereby generating a mouse line suitable for Cre-induced tissue-specific knockout experiments. Analysis of embryos carrying different combinations of these alleles revealed requirements for Fgf8 gene function during gastrulation, as well as cardiac, craniofacial, forebrain, midbrain and cerebellar development.

Disruption of human limb morphogenesis by a dominant negative mutation in CDMP1.

Chondrodysplasia Grebe type (CGT) is an autosomal recessive disorder characterized by severe limb shortening and dysmorphogenesis. We have identified a causative point mutation in the gene encoding the bone morphogenetic protein (BMP)-like molecule, cartilage-derived morphogenetic protein-1 (CDMP-1). The mutation substitutes a tyrosine for the first of seven highly conserved cysteine residues in the mature active domain of the protein. We demonstrate that the mutation results in a protein that is not secreted and is inactive in vitro. It produces a dominant negative effect by preventing the secretion of other, related BMP family members. We present evidence that this may occur through the formation of heterodimers. The mutation and its proposed mechanism of action provide the first human genetic indication that composite expression patterns of different BMPs dictate limb and digit morphogenesis.

GGF2 (Nrg1-ß3) treatment enhances NG2+ cell response and improves functional recovery after spinal cord injury.

The adult spinal cord contains a pool of endogenous glial precursor cells, which spontaneously respond to spinal cord injury (SCI) with increased proliferation. These include oligodendrocyte precursor cells that express the NG2 proteoglycan and can differentiate into mature oligodendrocytes. Thus, a potential approach for SCI treatment is to enhance the proliferation and differentiation of these cells to yield more functional mature glia and improve remyelination of surviving axons. We previously reported that soluble glial growth factor 2 (GGF2)- and basic fibroblast growth factor 2 (FGF2)-stimulated growth of NG2(+) cells purified from injured spinal cord in primary culture. This study examines the effects of systemic administration of GGF2 and/or FGF2 after standardized contusive SCI in vivo in both rat and mouse models. In Sprague-Dawley rats, 1 week of GGF2 administration, beginning 24 h after injury, enhanced NG2(+) cell proliferation, oligodendrogenesis, chronic white matter at the injury epicenter, and recovery of hind limb function. In 2',3'-cyclic-nucleotide 3'-phosphodiesterase-enhanced green fluorescent protein mice, GGF2 treatment resulted in increased oligodendrogenesis and improved functional recovery, as well as elevated expression of the stem cell transcription factor Sox2 by oligodendrocyte lineage cells. Although oligodendrocyte number was increased chronically after SCI in GGF2-treated mice, no evidence of increased white matter was detected. However, GGF2 treatment significantly increased levels of P0 protein-containing peripheral myelin, produced by Schwann cells that infiltrate the injured spinal cord. Our results suggest that GGF2 may have therapeutic potential for SCI by enhancing endogenous recovery processes in a clinically relevant time frame.

Transcriptome analysis reveals specific changes in osteoarthritis synovial fibroblasts.

OBJECTIVE: Changes in rheumatoid arthritis synovial fibroblast (RASF) gene expression are usually defined by a comparison to osteoarthritis synovial fibroblasts (OASFs). This study was undertaken to analyse the transcriptome of OASFs as compared to RASFs and healthy synovial fibroblasts (HSFs). METHODS: The authors used microarray messenger RNA expression profiling of synovial fibroblasts cultured from osteoarthritis (OA), rheumatoid arthritis and normal synovial tissues. Quantitative real-time PCR of selected genes was performed to validate microarray data. Analysis of variance, Student t test and the Benjamini-Hochberg multiple testing correction method for multiple testing correction were used to determine the statistical significance of the changes between the three groups. RESULTS: Larger numbers of transcripts showed a differential expression in OASFs versus the other groups, rather than in RASFs versus HSFs. Cluster analysis confirmed that the differences between the three groups were mostly due to the differences between OA and the other groups. Functional classification identified a significant number of genes related to growth factor activities, cell adhesion, neurotransmission and Ras signalling that are differentially expressed in OASFs. Classical proinflammatory factors or proteases involved in cartilage degradation were not found to be overexpressed in OASFs. CONCLUSION: Cultured OASFs display a more homogeneous transcriptomic profile than RASFs when compared to HSFs. This supports the participation of synovial fibroblasts in the pathogenesis of OA and may reflect global defects in the mesenchyma-derived lineages of the different tissues in OA joints. These data support individual heterogeneity among RASFs and advise against the use of OASFs as controls.

Telomerase modulates expression of growth-controlling genes and enhances cell proliferation.

Most somatic cells do not express sufficient amounts of telomerase to maintain a constant telomere length during cycles of chromosome replication. Consequently, there is a limit to the number of doublings somatic cells can undergo before telomere shortening triggers an irreversible state of cellular senescence. Ectopic expression of telomerase overcomes this limitation, and in conjunction with specific oncogenes can transform cells to a tumorigenic phenotype. However, recent studies have questioned whether the stabilization of chromosome ends entirely explains the ability of telomerase to promote tumorigenesis and have resulted in the hypothesis that telomerase has a second function that also supports cell division. Here we show that ectopic expression of telomerase in human mammary epithelial cells (HMECs) results in a diminished requirement for exogenous mitogens and that this correlates with telomerase-dependent induction of genes that promote cell growth. Furthermore, we show that inhibiting expression of one of these genes, the epidermal growth factor receptor (EGFR), reverses the enhanced proliferation caused by telomerase. We conclude that telomerase may affect proliferation of epithelial cells not only by stabilizing telomeres, but also by affecting the expression of growth-promoting genes.

Induction of activin A is essential for the neuroprotective action of basic fibroblast growth factor in vivo.

Exogenous application of neurotrophic growth factors has emerged as a new and particularly promising approach not only to promote functional recovery after acute brain injury but also to protect neurons against the immediate effect of the injury. Among the various growth factors and cytokines studied so far, the neuroprotective and neurotrophic profile of basic fibroblast growth factor (bFGF) is the best documented. Using an animal model of acute excitotoxic brain injury, we report here that the neuroprotective action of bFGF, which is now being tested in stroke patients, depends on the induction of activin A, a member of the transforming growth factor-beta superfamily. Our evidence for this previously unknown mechanism of action of bFGF is that bFGF strongly enhanced lesion-associated induction of activin A; in the presence of the activin-neutralizing protein follistatin, bFGF was no longer capable of rescuing neurons from excitotoxic death; and recombinant activin A exerted a neuroprotective effect by itself. Our data indicate that the development of substances influencing activin expression or receptor binding should offer new ways to fight neuronal loss in ischemic and traumatic brain injury.

Activin A/erythroid differentiation factor is induced during human monocyte activation.

Activin A/erythroid differentiation factor (EDF), a dimeric polypeptide hormone composed of two beta A subunits, regulates growth and erythroid differentiation of human hematopoietic progenitor and erythroleukemia cells. We have identified activated human peripheral blood monocytes as a natural source of activin A/EDF. In these cells, lipopolysaccharide (LPS) induced rapidly the expression of the beta A subunit mRNAs through protein kinase C-dependent transcriptional regulation. The beta A subunit mRNA expression was also increased by 1,25-dihydroxyvitamin D3, an inducer of macrophage maturation of monocytes. Western analysis with an anti-beta A antibody and an erythroid differentiation bioassay confirmed that the conditioned media of LPS-activated monocytes contained the activin A/EDF protein. We suggest that monocyte/macrophage-derived activin A/EDF may not only modulate hematopoiesis but may also act as a mediator molecule in the diverse physiologic and pathogenetic events in which these cells are involved.

Human T cell hybridomas secreting factors for IgA-specific help, polyclonal B cell activation, and B cell proliferation.

Human T-T hybridomas were established by fusion of concanavalin A-activated OKT-4+ T cells with hypoxanthine guanine phosphoribosyl transferase-deficient as well as nondeficient T cell lines. Four hybrids were selected for further study. Supernatant from hybrid clone J1.3 specifically enhanced IgA production and secretion by isolated human B cells, with increases in IgA plaque-forming cells approaching those seen with addition of autologous T cells and pokeweed mitogen. A monoclonal lymphocytic leukemia with membrane IgA also differentiated to IgA plasma cells by this supernatant. Evidence suggests that this hybrid supernatant acts on post-switch IgA-committed B cells. The other hybrids were not isotype specific; hybrid J2S1 enhanced polyclonal Ig secretion and hybrids K1 and K8 induced B cell proliferation without induction of Ig secretion.

B cell growth factors and B cell differentiation factor from human T hybridomas. Two distinct kinds of B cell growth factor and their synergism in B cell proliferation.

Human T hybridomas secreting B cell growth factors (BCGF) and B cell differentiation factor (BCDF) have been established. Hybrid clones 77-A, 94-C, and 98-F secreted BCGF that induced proliferation of anti-IgM-stimulated normal B cells. The culture supernatant from 77-A cells could also maintain continuous proliferation of colony-forming B cells, but the factor from 94-C could not. The addition of the supernatant from 94-C cells to that from 77-A cells, however, synergistically augmented the proliferation of colony-forming B cells, demonstrating the existence of two distinct kinds of BCGF and the synergism between them. These supernatants, however, showed no interleukin 2 (IL-2) or BCDF activity. A hybrid clone, 90-E, secreted BCDF. The culture supernatant induced Ig production in Cowan I-stimulated normal B cells or in a transformed B cell line, CESS. However, the supernatant had no BCGF or IL-2 activity. Anti-Ig-stimulated B cells, but not IL-2-dependent T cells, absorbed BCGF activity and CESS cells absorbed BCDF activity but not BCGF activity in the culture supernatants from T hybridomas. Taken collectively, the results demonstrated that IL-2, BCGF, and BCDF were different molecules and acceptors specific for the each molecule are present on the each target cell.

Viruses disrupt functions of human lymphocytes. II. Measles virus suppresses antibody production by acting on B lymphocytes.

Measles virus infection is associated with suppression of immune functions both in vivo and in vitro. The virus infects T lymphocytes, B lymphocytes, and monocytes, but does not produce cytolysis. One consequence of infection in vitro is the failure of T and B lymphocyte mixtures to cooperate in secreting Ig in a PWM-driven system. Here we report that this defect in Ig secretion resides in the infected B lymphocyte, but not in the T lymphocyte or monocyte. Further, NK cells are not involved, since neither their depletion nor reconstitution abrogates suppression of B cell function. Proliferation of B cells in the early culture period is suppressed, suggesting that measles virus suppresses B cell development at the activation or proliferation stages, but does not affect terminal differentiation into Ig secreting cells.

The in vitro generation and sustained culture of nude mouse cytolytic T-lymphocytes.

In addition to allowing for the long-term culture of both murine and human cytolytic T lymphocytes, T-cell growth factor (TCGF) functions as the key proliferation-inducing second signal in both T-cell antigen sensitization and mitogenesis. The observation that thymocytes responded normally to T-cell mitogens in the presence of TCGF, prompted the investigation of the effect of TCGF on nude mouse lymphocyte responses in vitro. We found that spleen, lymph node, and bone marrow cells, isolated from nude mice, were incapable of producing TCGF yet responded normally to T-cell mitogen sensitization provided stimulation was conducted in the presence of TCGF. Nude mouse spleen cells were also capable of responding to alloantigen sensitization in mixed lymphocyte cultures (NLMC) conducted in the presence of TCGF. Thy-1 antigen-positive cells harvested from TCGF-supplemented nude mouse MLC effectively mediated the cytolysis of alloantigen-specific target cells as tested in standard 51Cr-release assays. Cytolytic nude mouse effector cells have remained in TCGF-dependent culture for over 3 mo during which they have continued to mediate significant levels of alloantigen-specific cytolytic reactivity. These results suggest that prothymocytes present in nude mice are capable of responding to immunologic stimuli by differentiating, in vitro, into cytolytic T lymphocytes and that furthermore, a major function of the thymus may be to effect the maturation of TCGF-producing cells.

Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin.

We investigated the expression and distribution of keratinocyte growth factor (KGF) (FGF-7) and its receptor (KGFR) during reepithelialization of human skin. KGF mRNA levels increased rapidly by 8-10-fold and remained elevated for several days. In contrast, KGFR transcript levels decreased early but were significantly elevated by 8-9 d. A KGF-immunoglobulin G fusion protein (KGF-HFc), which specifically and sensitively detects the KGFR, localized the receptor to differentiating keratinocytes of control epidermis, but revealed a striking decrease in receptor protein expression during the intermediate period of reepithelization. Suramin, which blocked KGF binding and stripped already bound KGF from its receptor, failed to unmask KGFRs in tissue sections from the intermediate phase of wound repair. The absence of KGFR protein despite increased KGFR transcript levels implies functional receptor downregulation in the presence of increased KGF. This temporal modulation of KGF and KGFRs provides strong evidence for the functional involvement of KGF in human skin reepithelialization.

Expression of the chemokine N51/KC in the thymus and epidermis of transgenic mice results in marked infiltration of a single class of inflammatory cells.

Transgenic mice expressing the chemokine N51/KC in thymus, skin, and tongue showed a marked infiltration of a single class of inflammatory cells (neutrophils) in the sites of transgene expression. In the thymus, neutrophils were most numerous in the cortex and juxta-medullary regions, often forming aggregates or clusters. A similar, but less intense, neutrophilic infiltrate occurred in close proximity to the epidermal basal layer of the tongue and skin. No morphologic evidence of injury was observed in the thymus, skin, or tongue of these transgenic mice, indicating that N51/KC expression induces recruitment but not inflammatory activation of neutrophils. The lack of activation in the thymus resulted in a large senescent neutrophilic population that was phagocytosed by thymic macrophages and epithelial-reticular cells. These results indicate that N51/KC is a neutrophil chemoattractant in vivo and establish these transgenic mice as effective models to study the phenomena of recruitment and clearance of neutrophils, events that are critical for the initiation and resolution of the inflammatory response.

Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia.

Mucosal organs such as the intestine are supported by a rich and complex underlying vasculature. For this reason, the intestine, and particularly barrier-protective epithelial cells, are susceptible to damage related to diminished blood flow and concomitant tissue hypoxia. We sought to identify compensatory mechanisms that protect epithelial barrier during episodes of intestinal hypoxia. Initial studies examining T84 colonic epithelial cells revealed that barrier function is uniquely resistant to changes elicited by hypoxia. A search for intestinal-specific, barrier-protective factors revealed that the human intestinal trefoil factor (ITF) gene promoter bears a previously unappreciated binding site for hypoxia-inducible factor (HIF)-1. Hypoxia resulted in parallel induction of ITF mRNA and protein. Electrophoretic mobility shift assay analysis using ITF-specific, HIF-1 consensus motifs resulted in a hypoxia-inducible DNA binding activity, and loading cells with antisense oligonucleotides directed against the alpha chain of HIF-1 resulted in a loss of ITF hypoxia inducibility. Moreover, addition of anti-ITF antibody resulted in a loss of barrier function in epithelial cells exposed to hypoxia, and the addition of recombinant human ITF to vascular endothelial cells partially protected endothelial cells from hypoxia-elicited barrier disruption. Extensions of these studies in vivo revealed prominent hypoxia-elicited increases in intestinal permeability in ITF null mice. HIF-1-dependent induction of ITF may provide an adaptive link for maintenance of barrier function during hypoxia.

Immune regulation of in vitro murine megakaryocyte development. Role of T lymphocytes and Ia antigen expression.

Mitogen-activated murine T lymphocytes or T cell hybridomas produce an activity (megakaryocyte [Mk] potentiator activity) that enhances the in vitro growth and development of Mk colonies. This activity was found in optimal concentrations (2.5%) in T cell hybridoma-conditioned medium, and was also produced by feeder layers of concanavalin A-activated T cells. A subpopulation of murine Mk progenitor cells (colony-forming units; CFU-Mk) bears the Ia antigen. Separate experiments indicated that T cell products stimulate CFU-Mk by increasing their basal levels of Ia expression as well as the frequency of cells actively synthesizing DNA. The hypothesis that the expression of this antigen was related to the cell cycle status of these progenitor cells was confirmed in studies that indicated that ablation of actively cycling cells in vivo abrogated the cytotoxic effects of anti-Ia monoclonal antibodies. The interdependence of T cell lymphokine regulation of both Ia expression and cell cycle status was also seen in in vitro experiments in which Ia+ progenitor cells were eliminated by complement-dependent cytotoxicity. The removal of Ia+ cells prevented 5-hydroxyurea-mediated inhibition of cells in S phase. We hypothesize that immune modulation of megakaryocytopoiesis occurs via soluble T cell products that augment Mk differentiation. Further, the mechanism of immune recognition/modulation may occur via Ia antigens present on the surface of these progenitor cells.

Identification of a B cell differentiation factor(s) spontaneously produced by proliferating T cells in murine lupus strains of the lpr/lpr genotype.

Lymph node and spleen cells of the autoimmune MRL/Mp-lpr/lpr mouse strain spontaneously produce (in the absence of mitogenic stimulation) a factor(s) that induces B cell differentiation. This factor is not produced by the congenic MRL/n mouse strain that lacks the lpr gene or by normal mouse strains. However, lymphoid cells of the B6-lpr/lpr (B6/1) strain also produce a B cell differentiation factor. Although the factor acts on resting B cells, its effect is greatly magnified by activating the B cells with anti-mu or lipopolysaccharide. MRL/l mice begin producing the factor as early as 1 mo of age but levels increase with age and appearance of lymphoproliferation. Cell depletion studies reveal that this factor is produced by T cells of the Lyt-1+2-phenotype. Because of its association with the lpr/lpr genotype, we term this B cell differentiation factor L-BCDF. Functional analysis of L-BCDF reveals that it acts regardless of cell density in culture and in the absence of interleukin 2 (IL-2). In fact, the increase in the production of L-BCDF by MRL/1 T cells with aging occurs concomitantly with a marked decrease in their ability to produce IL-2. No T cell replacing factor activity or B cell growth factor-like activity can be detected in MRL/l-derived supernatants. L-BCDF induces both IgM and IgG synthesis in lipopolysaccharide-activated B cells; however, it has a greater effect on IgG secretion. In particular, the production of IgG1, IgG2a, and IgG2b are markedly enhanced in the presence of L-BCDF. The spontaneous production of L-BCDF by T cells of SLE mice of lpr/lpr genotype suggests an association of this factor with autoimmunity.

Regulation of parasite-induced eosinophilia: selectively increased interleukin 5 production in helminth-infected patients.

Production of the eosinophilogenic cytokines interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and IL-5 by mitogen-stimulated peripheral blood mononuclear cells was compared between 11 noneosinophilic individuals and seven patients with helminth-induced eosinophilia. Both the kinetics and quantities of IL-3 and GM-CSF were similar in the two groups. In contrast, IL-5 production at both the protein and the mRNA level was markedly greater in the eosinophilic patients, an observation suggesting that IL-5 may be particularly important in mediating the selective eosinophilia seen in filarial and other helminth infections.

A distinct wave of human T cell receptor gamma/delta lymphocytes in the early fetal thymus: evidence for controlled gene rearrangement and cytokine production.

The rearrangement and expression of human T cell receptor (TCR)-gamma and -delta gene segments in clonal and polyclonal populations of early fetal and postnatal human TCR-gamma/delta thymocytes were examined. The data suggest that the TCR-gamma and -delta loci rearrange in an ordered and coordinated fashion. Initial rearrangements at the TCR-delta locus join V delta 2 to D delta 3, and initial rearrangements at the TCR-gamma locus join downstream V gamma gene segments (V gamma 1.8 and V gamma 2) to upstream J gamma gene segments associated with C gamma 1. These rearrangements are characterized by minimal junctional diversity. At later times there is a switch at the TCR-delta locus such that V delta 1 is joined to upstream D delta gene segments, and a switch at the TCR-gamma locus such that upstream V gamma gene segments are joined to downstream J gamma gene segments associated with C gamma 2. These rearrangements are characterized by extensive junctional diversity. Programmed rearrangement explains in part the origin of discrete subpopulations of peripheral blood TCR-gamma/delta lymphocytes that have been defined in previous studies. In addition, cytokine production by early fetal and postnatal TCR-gamma/delta thymocyte clones was examined. Fetal thymocyte clones produced significant levels of IL-4 and IL-5 following stimulation, whereas postnatal thymocyte clones did not produce these cytokines. Thus, these cell populations may represent functionally distinct subsets as well.