Peptide-based immunotherapy in lupus: Where are we now?

In autoimmune rheumatic diseases, immune hyperactivity and chronic inflammation associate with immune dysregulation and the breakdown of immune self-tolerance. A continued, unresolved imbalance between effector and regulatory immune responses further exacerbates inflammation that ultimately causes tissue and organ damage. Many treatment modalities have been developed to restore the immune tolerance and immmunoregulatory balance in autoimmune rheumatic diseases, including the use of peptide-based therapeutics or the use of nanoparticles-based nanotechnology. This review summarizes the state-of-the-art therapeutic use of peptide-based therapies in autoimmune rheumatic diseases, with a specific focus on lupus.

Synthetically glycosylated antigens for the antigen-specific suppression of established immune responses.

Inducing antigen-specific tolerance during an established immune response typically requires non-specific immunosuppressive signalling molecules. Hence, standard treatments for autoimmunity trigger global immunosuppression. Here we show that established antigen-specific responses in effector T cells and memory T cells can be suppressed by a polymer glycosylated with N-acetylgalactosamine (pGal) and conjugated to the antigen via a self-immolative linker that allows for the dissociation of the antigen on endocytosis and its presentation in the immunoregulatory environment. We show that pGal-antigen therapy induces antigen-specific tolerance in a mouse model of experimental autoimmune encephalomyelitis (with programmed cell-death-1 and the co-inhibitory ligand CD276 driving the tolerogenic responses), as well as the suppression of antigen-specific responses to vaccination against a DNA-based simian immunodeficiency virus in non-human primates. Our findings show that pGal-antigen therapy invokes mechanisms of immune tolerance to resolve antigen-specific inflammatory T-cell responses and suggest that the therapy may be applicable across autoimmune diseases.

Identification and Contribution of Inflammation-Induced Novel MicroRNA in the Pathogenesis of Systemic Lupus Erythematosus.

Recently microRNAs (miRNAs) have been recognized as powerful regulators of many genes and pathways involved in the pathogenesis of inflammatory diseases including Systemic Lupus Erythematosus (SLE). SLE is an autoimmune disease characterized by production of various autoantibodies, inflammatory immune cells, and dysregulation of epigenetic changes. Several candidate miRNAs regulating inflammation and autoimmunity in SLE are described. In this study, we found significant increases in the expression of miR21, miR25, and miR186 in peripheral blood mononuclear cells (PBMCs) of SLE patients compared to healthy controls. However, miR146a was significantly decreased in SLE patients compared to healthy controls and was negatively correlated with plasma estradiol levels and with SLE disease activity scores (SLEDAI). We also found that protein levels of IL-12 and IL-21 were significantly increased in SLE patients as compared to healthy controls. Further, our data shows that protein levels of IL-12 were positively correlated with miR21 expression and protein levels of IL-21 positively correlated with miR25 and miR186 expression in SLE patients. In addition, we found that levels of miR21, miR25, and miR186 positively correlated with SLEDAI and miR146a was negatively correlated in SLE patients. Thus, our data shows a dynamic interplay between disease pathogenesis and miRNA expression. This study has translational potential and may identify novel therapeutic targets in patients with SLE.

Cellular and Molecular Phenotypes of pConsensus Peptide (pCons) Induced CD8+ and CD4+ Regulatory T Cells in Lupus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with widespread inflammation, immune dysregulation, and is associated with the generation of destructive anti-DNA autoantibodies. We have shown previously the immune modulatory properties of pCons peptide in the induction of both CD4+ and CD8+ regulatory T cells which can in turn suppress development of the autoimmune disease in (NZB/NZW) F1 (BWF1) mice, an established model of lupus. In the present study, we add novel protein information and further demonstrate the molecular and cellular phenotypes of pCons-induced CD4+ and CD8+ Treg subsets. Flow cytometry analyses revealed that pCons induced CD8+ Treg cells with the following cell surface molecules: CD25highCD28high and low subsets (shown earlier), CD62Lhigh, CD122low, PD1low, CTLA4low, CCR7low and 41BBhigh. Quantitative real-time PCR (qRT-PCR) gene expression analyses revealed that pCons-induced CD8+ Treg cells downregulated the following several genes: Regulator of G protein signaling (RGS2), RGS16, RGS17, BAX, GPT2, PDE3b, GADD45ß and programmed cell death 1 (PD1). Further, we confirmed the down regulation of these genes by Western blot analyses at the protein level. To our translational significance, we showed herein that pCons significantly increased the percentage of CD8+FoxP3+ T cells and further increased the mean fluorescence intensity (MFI) of FoxP3 when healthy peripheral blood mononuclear cells (PBMCs) are treated with pCons (10 µg/ml, for 24-48 hours). In addition, we found that pCons reduced apoptosis in CD4+ and CD8+ T cells and B220+ B cells of BWF1 lupus mice. These data suggest that pCons stimulates cellular, immunological, and molecular changes in regulatory T cells which in turn protect against SLE autoimmunity.

Interferon Genes Are Influenced by 17ß-Estradiol in SLE.

Recent evidence suggests the existence of a nexus between inflammatory pathways and the female sex hormone 17ß-estradiol, resulting in increased interferon-stimulated genes (ISGs), autoantibodies, and dysregulation of immune cells in SLE. However, the molecular mechanisms and the effect of estradiol on candidate target genes and their pathways remains poorly understood. Our previous work suggests that female SLE patients have increased estradiol levels compared to healthy controls. In the present study, we explored the effects of 17ß-estradiol treatment on expression of IFN (interferons)-stimulated genes and pro-inflammatory cytokines/chemokines. We found significantly increased (5-10-fold) expression of IFN-regulated genes in healthy females. Furthermore, we found significantly increased plasma levels of IL-6, IL-12, IL-17, IL-18, stem cell factor (SCF), and IL-21/IL-23 in SLE patients compared to healthy controls, and those levels positively correlated with the plasma levels of 17ß-estradiol. In addition, levels of IL-21 positively correlated with the SLE disease activity index (SLEDAI) score of SLE patients. In vitro treatment of PBMCs from either SLE patients or healthy controls with 17ß-estradiol at physiological concentration (~50 pg/ml) also significantly increased secretion of many pro-inflammatory cytokines and chemokines (IL-6, IL-12, IL-17, IL-8, IFN-γ; MIP1α, and MIP1ß) in both groups. Further our data revealed that 17ß-estradiol significantly increased the percentage of CD3+CD69+ and CD3+IFNγ+ T cells; whereas, simultaneous addition of 17ß-estradiol and an ERα inhibitor prevented this effect. Collectively, our findings indicate that 17ß-estradiol participates in the induction of pro-inflammatory cytokines and chemokines and further influences interferon genes and pathways.

Effects of Peptide-Induced Immune Tolerance on Murine Lupus.

The regulation of autoimmunity and the molecular mechanisms by which different immune cells, including T cells, polymorphonuclear leukocytes (PMN-granulocytes), and B cells suppress autoimmune diseases is complex. We have shown previously that BWF1 lupus mice are protected from autoimmunity after i.v. injection or oral administration of tolerogenic doses of pCons, an artificial synthetic peptide based on sequences containing MHC class I and MHC class II determinants in the VH region of a J558-encoded BWF1 anti-DNA Ab. Several T cell subsets can transfer this tolerance. In this study, we determined the potential roles of granulocytes, B cells and regulatory T cells altered by pCons treatment in the BWF1 (NZB/NZW) mouse model of lupus. Immunophenotyping studies indicated that pCons treatment of BWF1 mice significantly increased CD4+FoxP3+ T cells, reduced the percent of B cells expressing CD19+CD5+ but increased the percent of CD19+CD1d+ regulatory B cells and increased the ability of the whole B cell population to suppress IgG anti-DNA production in vitro. pCons treatment significantly decreased the expression of CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) in CD8+ T cells. In addition, peptide administration modified granulocytes so they became suppressive. We co-cultured sorted naïve B cells from mice making anti-DNA Ab (supported by addition of sorted naive CD4+ and CD8+ T cells from young auto-antibody-negative BWF1 mice) with sorted B cells or granulocytes from tolerized mice. Both tolerized granulocytes and tolerized B cells significantly suppressed the production of anti-DNA in vitro. In granulocytes from tolerized mice compared to saline-treated littermate controls, real-time PCR analysis indicated that expression of interferon-induced TNFAIP2 increased more than 2-fold while Ptdss2 and GATA1 mRNA were up-regulated more than 10-fold. In contrast, expression of these genes was significantly down-regulated in tolerized B cells. Further, another IFN-induced protein, Bcl2, was reduced in tolerized B cells as determined by Western blot analyses. In contrast, expression of FoxP3 was significantly increased in tolerized B cells. Together, these data suggest that B cells and granulocytes are altered toward suppressive functions by in vivo tolerization of BWF1 mice with pCons and it is possible these cell types participate in the clinical benefits seen in vivo.

Sex Hormones and Gender Influence the Expression of Markers of Regulatory T Cells in SLE Patients.

Regulatory T cells have been implicated in the regulation and maintenance of immune homeostasis. Whether gender and sex hormones differentially influence the expression and function of regulatory T cell phenotype and their influence on FoxP3 expression remains obscure. We provide evidence in this study that the number and percent of human regulatory T cells (Tregs) expressing CD4+ and CD8+ are significantly reduced in healthy females compared to healthy males. In addition, both CD4+CD25+hi and CD8+CD25+hi subsets in healthy males have a 2-3 fold increase in FoxP3 mRNA expression compared to healthy females. Female SLE patients, compared to healthy women, have elevated plasma levels of estradiol and decreased levels of testosterone. Higher levels of testosterone correlate with higher expression of FoxP3 in CD4+CD25hiCD127low putative Tregs in women with SLE. Incubation of CD4+ regulatory T cells with 17ß-estradiol at physiological levels generally decreased FoxP3 expression in females with SLE. These data suggest that females may be more susceptible than males to SLE and other autoimmune diseases in part because they have fewer Tregs and reduced FoxP3 expression within those cells due to normal E2 levels which suppress FoxP3 expression. In addition, low levels of plasma testosterone in women may further reduce the ability of the Tregs to express FoxP3. These data suggest that gender and sex hormones can influence susceptibility to SLE via effects on regulatory T cells and FoxP3 expression.

CD8+ T regulatory cells in lupus.

T regulatory cells (Tregs) have a key role in the maintenance of immune homeostasis and the regulation of immune tolerance by preventing the inflammation and suppressing the autoimmune responses. Numerical and functional deficits of these cells have been reported in systemic lupus erythematosus (SLE) patients and mouse models of SLE, where their imbalance and dysregulated activities have been reported to significantly influence the disease pathogenesis, progression and outcomes. Most studies in SLE have focused on CD4+ Tregs and it has become clear that a critical role in the control of immune tolerance after the breakdown of self-tolerance is provided by CD8+ Tregs. Here we review the role, cellular and molecular phenotypes, and mechanisms of action of CD8+ Tregs in SLE, including ways to induce these cells for immunotherapeutic modulation in SLE.

Osteoprotegerin reduces osteoclast resorption activity without affecting osteogenesis on nanoparticulate mineralized collagen scaffolds.

The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.

Nanoparticulate mineralized collagen glycosaminoglycan materials directly and indirectly inhibit osteoclastogenesis and osteoclast activation.

The ability of the extracellular matrix (ECM) to direct cell fate has generated the potential for developing a materials-only strategy for tissue regeneration. Previously, we described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material that efficiently induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without exogenous growth factors or progenitor cell expansion. In this work, we evaluated the interactions between MC-GAG and primary human osteoclasts (hOCs). In the absence of hMSCs, mineralized Col-GAG materials directly inhibited hOC viability, proliferation, and resorption in contrast to nonmineralized Col-GAG, which demonstrated a modest inhibition of resorptive activity only. Cocultures containing differentiating hMSCs with hOCs demonstrated increased hOC-mediated resorption only on Col-GAG while MC-GAG cocultures continued to inhibit resorption. Unlike Col-GAG, hMSCs on MC-GAG expressed increased amounts of osteoprotegerin (OPG) protein, the major endogenous osteoclast inhibitor. Interestingly, OPG expression was found to be antagonized by small mothers against decapentaplegic1/5 (Smad1/5) phosphorylation, an obligate pathway for osteogenic differentiation of hMSCs on MC-GAG, and potentiated by extracellular signal-regulated kinase (ERK1/2) phosphorylation. Collectively, these results suggested that the MC-GAG material both directly inhibited the osteoclast viability, proliferation, and resorptive activity as well as induced hMSCs to secrete osteoprotegerin, an antiosteoclastogenic factor, via a signalling pathway distinct from osteogenic differentiation.

Engineering advanced neural tissue constructs to mitigate acute cerebral inflammation after brain transplantation in rats.

Stroke, traumatic brain injuries, and other similar conditions often lead to significant loss of functional brain tissue and associated disruption of neuronal signaling. A common strategy for replacing lost neurons is the injection of dissociated neural stem cells or differentiated neurons. However, this method is unlikely to be suitable for replacing large brain cavities, and the resulting distribution of neurons may lack the necessary architecture to support appropriate brain function. Engineered neural tissues may be a viable alternative. Cell death is a prominent concern in neuronal grafting studies, a problem that could be magnified with the transplantation of engineered neural tissues. Here, we examined the effect of one contributor to cell death, acute cerebral inflammation, on neuronal survival after the transplantation of bioengineered constructs based on silk scaffolds. We found evidence of a high degree of inflammation and poor neuronal survival after introducing engineered constructs into the motor cortex of rats. Integrating a corticosteroid (methylprednisolone) into the constructs resulted in significantly improved neuron survival during the acute phase of inflammation. The improved construct survival was associated with decreased markers of inflammation and an anti-inflammatory state of the immune system due to the steroid treatment.

Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells.

The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, a correlation was described between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of small mothers against decapentaplegic ( Smad1/5) in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished extracellular signal regulated kinase 1/2 (ERK1/2) activation when compared to nonmineralized collagen glycosaminoglycan scaffolds (Col-GAG). This work utilizes a canonical BMPR inhibitor (dorsomorphin homologue 1, DMH1) and an inhibitor of the mitogen activated protein kinase/ERK kinase (MEK)/(ERK) cascade (PD98059) to characterize the necessity of each pathway for osteogenesis. While DMH1 inhibits runt-related transcription factor 2 (Runx2) and bone sialoprotein II (BSPII) gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG, PD98059 inhibits BSPII expression on Col-GAG independent of Runx2 expression. DMH1 inhibits mineralization on both Col-GAG and MC-GAG, however, PD98059 only inhibits mineralization on Col-GAG. DMH1 inhibits both Smad1/5 phosphorylation and Runx2 protein expression, whereas PD98059 inhibits ERK1/2 and c-Jun amino-terminal kinase 1/2 (JNK1/2) phosphorylation without affecting Runx2. Thus, activation of the canonical BMPR signaling is necessary for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Metabolic relationship between diabetes and Alzheimer's Disease affected by Cyclo(His-Pro) plus zinc treatment.

BACKGROUND: Association of Alzheimer's Disease (AD) with Type 2 Diabetes (T2D) has been well established. Cyclo(His-Pro) plus zinc (Cyclo-Z) treatment ameliorated diabetes in rats and similar improvements have been seen in human patients. Treatment of amyloid precursor protein (APP) transgenic mice with Cyclo-Z exhibited memory improvements and significantly reduced Aß-40 and Aß-42 protein levels in the brain tissues of the mice. SCOPE OF REVIEW: Metabolic relationship between AD and T2D will be described with particular attention to insulin sensitivity and Aß degradation in brain and plasma tissues. Mechanistic effect of insulin degrading enzyme (IDE) in decreasing blood glucose and brain Aß levels will be elucidated. Cyclo-Z effects on these biochemical parameters will be discussed. MAJOR CONCLUSION: Stimulation of IDE synthesis is effective for the clinical treatment of metabolic diseases including AD and T2D. GENERAL SIGNIFICANCE: Cyclo-Z might be the effective treatment of AD and T2D by stimulating IDE synthesis.

Nanoparticulate Mineralized Collagen Scaffolds and BMP-9 Induce a Long-Term Bone Cartilage Construct in Human Mesenchymal Stem Cells.

Engineering the osteochondral junction requires fabrication of a microenvironment that supports both osteogenesis and chondrogenesis. Multiphasic scaffold strategies utilizing a combination of soluble factors and extracellular matrix components are ideally suited for such applications. In this work, the contribution of an osteogenic nanoparticulate mineralized glycosaminoglycan scaffold (MC-GAG) and a dually chondrogenic and osteogenic growth factor, BMP-9, in the differentiation of primary human mesenchymal stem cells (hMSCs) is evaluated. Although 2D cultures demonstrate alkaline phosphatase activity and mineralization of hMSCs induced by BMP-9, MC-GAG scaffolds do not demonstrate significant differences in the collagen I expression, osteopontin expression, or mineralization. Instead, BMP-9 increases expression of collagen II, Sox9, aggrecan (ACAN), and cartilage oligomeric protein. However, the hypertrophic chondrocyte marker, collagen X, is not elevated with BMP-9 treatment. In addition, histologic analyses demonstrate that while BMP-9 does not increase mineralization, BMP-9 treatment results in an increase of sulfated glycosaminoglycans. Thus, the combination of BMP-9 and MC-GAG stimulates chondrocytic and osteogenic differentiation of hMSCs.

Nanoparticulate mineralized collagen scaffolds induce in vivo bone regeneration independent of progenitor cell loading or exogenous growth factor stimulation.

Current strategies for skeletal regeneration often require co-delivery of scaffold technologies, growth factors, and cellular material. However, isolation and expansion of stem cells can be time consuming, costly, and requires an additional procedure for harvest. Further, the introduction of supraphysiologic doses of growth factors may result in untoward clinical side effects, warranting pursuit of alternative methods for stimulating osteogenesis. In this work, we describe a nanoparticulate mineralized collagen glycosaminoglycan scaffold that induces healing of critical-sized rabbit cranial defects without addition of expanded stem cells or exogenous growth factors. We demonstrate that the mechanism of osteogenic induction corresponds to an increase in canonical BMP receptor signalling secondary to autogenous production of BMP-2 and -9 early and BMP-4 later during differentiation. Thus, nanoparticulate mineralized collagen glycosaminoglycan scaffolds may provide a novel growth factor-free and ex vivo progenitor cell culture-free implantable method for bone regeneration.

Micro-CT in the Assessment of Pediatric Renal Osteodystrophy by Bone Histomorphometry.

BACKGROUND AND OBJECTIVES: Computed tomography (CT) measurements can distinguish between cortical and trabecular bone density in vivo. High-resolution CTs assess both bone volume and density in the same compartment, thus potentially yielding information regarding bone mineralization as well. The relationship between bone histomorphometric parameters of skeletal mineralization and bone density from microcomputed tomography (µCT) measurements of bone cores from patients on dialysis has not been assessed. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Bone cores from 68 patients with ESRD (age =13.9±0.5 years old; 50% men) and 14 controls (age =15.3±3.8 years old; 50% men) obtained as part of research protocols between 1983 and 2006 were analyzed by bone histomorphometry and µCT. RESULTS: Bone histomorphometric diagnoses in the patients were normal to high bone turnover in 76%, adynamic bone in 13%, and osteomalacia in 11%. Bone formation rate did not correlate with any µCT determinations. Bone volume measurements were highly correlated between bone histomorphometry and µCT (bone volume/tissue volume between the two techniques: r=0.70; P<0.001, trabecular thickness and trabecular separation: r=0.71; P<0.001, and r=0.56; P<0.001, respectively). Osteoid accumulation as determined by bone histomorphometry correlated inversely with bone mineral density as assessed by µCT (osteoid thickness: r=-0.32; P=0.01 and osteoid volume: r=-0.28; P=0.05). By multivariable analysis, the combination of bone mineral density and bone volume (as assessed by µCT) along with parathyroid hormone and calcium levels accounted for 38% of the variability in osteoid volume (by histomorphometry). CONCLUSIONS: Measures of bone volume can be accurately assessed with µCT. Bone mineral density is lower in patients with excessive osteoid accumulation and higher in patients with adynamic, well mineralized bone. Thus, bone mineralization may be accurately assessed by µCT of bone biopsy cores. Additional studies are warranted to define the value of high-resolution CT in the prediction of bone mineralization in vivo.

Optimizing Collagen Scaffolds for Bone Engineering: Effects of Cross-linking and Mineral Content on Structural Contraction and Osteogenesis.

INTRODUCTION: Osseous defects of the craniofacial skeleton occur frequently in congenital, posttraumatic, and postoncologic deformities. The field of scaffold-based bone engineering emerged to address the limitations of using autologous bone for reconstruction of such circumstances. In this work, the authors evaluate 2 modifications of three-dimensional collagen-glycosaminoglycan scaffolds in an effort to optimize structural integrity and osteogenic induction. METHODS: Human mesenchymal stem cells (hMSCs) were cultured in osteogenic media on nonmineralized collagen-glycosaminoglycan (C-GAG) and nanoparticulate mineralized collagen-glycosaminoglycan (MC-GAG) type I scaffolds, in the absence and presence of cross-linking. At 1, 7, and 14 days, mRNA expression was analyzed using quantitative real-time -reverse-transcriptase polymerase chain reaction for osteocalcin (OCN) and bone sialoprotein (BSP). Structural contraction was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization was detected by microcomputed tomographic (micro-CT) imaging at 8 weeks. Statistical analyses were performed with Student t-test. RESULTS: Nanoparticulate mineralization of collagen-glycosaminoglycan scaffolds increased expression of both OCN and BSP. Cross-linking of both C-GAG and MC-GAG resulted in decreased osteogenic gene expression; however, structural contraction was significantly decreased after cross-linking. Human mesenchymal stem cells-directed mineralization, detected by micro-CT, was increased in nanoparticulate mineralized scaffolds, although the density of mineralization was decreased in the presence of cross-linking. CONCLUSIONS: Optimization of scaffold material is an essential component of moving toward clinically translatable engineered bone. Our current study demonstrates that the combination of nanoparticulate mineralization and chemical cross-linking of C-GAG scaffolds generates a highly osteogenic and structurally stable scaffold.

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway.

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling.

AIM: To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). METHODS: CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. RESULTS: CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels. CONCLUSION: CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways.

Constitutive expression of human telomerase enhances the proliferation potential of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are highly desirable cells for bone engineering due to the inherent multipotent nature of the cells. Unfortunately, there is a high degree of variability, as primary hMSC cultures quickly undergo replicative senescence with loss of proliferative potential as they are continually propagated in cell culture. We sought to reduce the variability of these cells by insertion and expression of human telomerase reverse transcriptase (TERT) to immortalize the cell line. hMSCs were transduced with a lentivirus containing the human TERT gene. The resulting cell line has been propagated through more than 70 population-doubling level (PDL) to date and continues to grow exhibiting the characteristic fibroblastic hMSC phenotype. Expression of TERT mRNA and protein activity was confirmed in the TERT-transduced cells. Mock-transduced hMSCs had almost undetectable levels of TERT mRNA and protein activity and lost proliferation potential at PDL 14. The enhanced growth capacity of the hMSC TERT cells was due to increased cell proliferation and reduced cellular senescence rather than due to inhibition of apoptosis. The multipotent nature of the TERT cells was confirmed by differentiation toward the osteoblastic and adipogenic lineages in vitro. Osteoblastic differentiation was confirmed by both expression of alkaline phosphate and mineral deposition visualized by Alizarin Red staining. Adipogenic differentiation was confirmed by production of lipid droplets, which were detected by Oil Red-O staining. In summary, we have generated a stable hMSC line that can be continually propagated and retains both osteoblastic and adipogenic differentiation potential.