CircPDE7B/miR-661 axis accelerates the progression of human keloid fibroblasts by upregulating fibroblast growth factor 2 (FGF2).

Circular RNAs (circRNAs) are implicated in keloidogenesis and development. We aimed to investigate the role of a new identified phosphodiesterase 7B-derived circRNA (hsa_circ_0002198; henceforth named as PDE7B) in human keloid fibroblasts (HKFs) and to further confirm its mechanism via competing endogenous RNA (ceRNA) network. Transcriptional and translational levels of circPDE7B, microRNA (miR)-661, fibroblast growth factor 2 (FGF2), cleaved caspase3, B-cell lymphoma (bcl)-2, and bcl-2-associated X protein (bax) were detected by real-time quantitative PCR and western blotting. Relationship among circPDE7B, miR-661, and FGF2 was confirmed by bioinformatics algorithm, dual-luciferase reporter assay, RNA immunoprecipitation, RNA pull-down assay, and Spearman's rank correlation analysis. Cell progression was measured by cell counting kit-8 assay, 5-ethynyl-2-deoxyuridine assay, transwell assays, and flow cytometry. Expression of circPDE7B was upregulated in human keloid tissues and HKFs, accompanied with miR-661 downregulation and FGF2 upregulation. High circPDE7B accelerated proliferation, migration, and invasion, and inhibited apoptosis. These effects were paralleled with increased bcl-2 and decreased cleaved caspase3 and bax. Moreover, low circPDE7B played opposite effects to high circPDE7B. Restoring miR-661 could suppress HKFs progression, while blocking miR-661 could facilitate that. Notably, miR-661 was directly sponged by circPDE7B and then directly governed FGF2 gene expression. Deleting miR-661 and re-expressing FGF2 both abrogated the suppression of circPDE7B knockdown in HKFs progression. In conclusion, circPDE7B might contribute to HKFs progression via functioning as ceRNA for miR-661, suggesting a novel circPDE7B/miR-661/FGF2 pathway underlying keloid formation and treatment.

Recombinant protein production-associated metabolic burden reflects anabolic constraints and reveals similarities to a carbon overfeeding response.

A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.

HOXD-AS1 facilitates cell migration and invasion as an oncogenic lncRNA by competitively binding to miR-877-3p and upregulating FGF2 in human cervical cancer.

BACKGROUND: Long non-coding RNAs (LncRNAs) are dysregulated in multiple human cancers and they are highly involved in tumor progression. Previous studies have identified the oncogenic lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) in human cancers, while its roles in cervical cancer (CC) remain unclear. Herein we intended to characterize the implication of HOXD-AS1 in CC. METHODS: qRT-PCR was applied to examine the relative expression of HOXD-AS1 in CC tissues, cell lines and transfected cells. Wound healing and transwell assays were applied to detect cell migration and invasion alteration. The targeting relationship between miRNA and mRNA/lncRNA was determined by dual luciferase reporter, qRT-PCR and western blot assays. RESULTS: HOXD-AS1 was overexpressed in CC tissues and cell lines. Its higher level predicted worse prognosis of CC patients. SiRNA mediated knockdown of HOXD-AS1 repressed CC cell migration and invasion, and its overexpression did the opposite. Mechanistically, HOXD-AS1 acted as a competing endogenous RNA (ceRNA) to sponge miR-877-3p and led to upregulation of FGF2, a target of miR-877-3p. Importantly, either miR-877-3p overexpression or FGF2 inhibition could abolish the migration and invasion promotion induced by HOXD-AS1. CONCLUSION: HOXD-AS1 functions as a tumor-promoting lncRNA via the miR-877-3p/FGF2 axis in CC. HOXD-AS1 might be a promising therapeutic target as well as a novel prognostic biomarker for CC.

Enhancement of cell proliferation and motility of mammalian cells grown in co-culture with Pichia pastoris expressing recombinant human FGF-2.

Many studies examining the biological function of recombinant proteins and their effects on the physiology of mammalian cells stipulate that the proteins be purified before being used as therapeutic agents. In this study, we explored the possibility of using unpurified recombinant proteins to treat mammalian cells. The recombinant protein was used directly from the expression source and the biological function was compared to purified commercially available, equivalent protein. The model for this purpose was recombinant FGF-2, expressed by Pichia pastoris, which was used to treat the murine fibroblast cell line, NIH/3T3. We generated a P. pastoris strain (yHL11) that constitutively secreted a biologically active recombinant FGF-2 protein containing an N-terminal c-myc epitope (Myc-FGF-2). Myc-FGF-2 was then used without purification either a) in the form of conditioned mammalian cell culture medium or b) during co-cultures of yHL11 with NIH/3T3 to induce higher proliferation and motility of NIH/3T3 cells. The effects of Myc-FGF-2 on cell physiology were comparable to commercially available FGF-2. To our knowledge, this is the first time the physiology of cultured mammalian cells had been successfully altered with a recombinant protein secreted by P. pastoris while the two species shared the same medium and culture conditions. Our data demonstrated the biological activity of unpurified recombinant FGF-2 on NIH/3T3 cells and provided a foundation for directly using unpurified recombinant proteins expressed by P. pastoris with mammalian cells, potentially as wound-healing therapeutics.

Fermentation, purification and immunogenicity of a recombinant tumor multi-epitope vaccine, VBP3.

The recombinant multi-epitope vaccine called VBP3 is designed to suppress tumor growth and angiogenesis through targeting both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA). We are aiming to produce VBP3 vaccine in a large scale and provide sufficient protein for pre-clinical study. High cost and potential toxicity are severe limitations of IPTG and we investigated whether lactose can mediate VBP3 induction. Firstly, we identified the biological characteristics and established a culture bank of VBP3 strains. The best-performing strains were selected and the fermentation mode of medium, bacterial growth and protein expression were optimized in shake flasks. We scaled up the VBP3 production in 10 L bioreactor using lactose as inducer and the protein yield was comparable with IPTG induction. Next, the target protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, with a SDS-PAGE purity over 90%. Further, the purified VBP3 vaccine was subcutaneously injected in BALB/c mice and elicited high-titer anti-bFGF (1:32,000) and anti-VEGFA (1:4000) antibodies. Take together, lactose was an applicable inducer for VBP3 production and the eligible product of VBP3 was harvested in the large-scale fermentation, supporting the industrial production and pre-clinical study in the future. The VBP3 vaccine with superior immunogenicity might be used as a potential therapeutic vaccine for tumor treatment.

circRAD18 sponges miR-208a/3164 to promote triple-negative breast cancer progression through regulating IGF1 and FGF2 expression.

As a new rising star of non-coding RNA, circular RNAs (circRNAs) emerged as vital regulators with biological functions in diverse of cancers. However, the function and precise mechanism of the vast majority of circRNAs in triple-negative breast cancer (TNBC) occurrence and progression have not been clearly elucidated. In the current study, we identified and further investigated hsa_circ_0002453 (circRAD18) by analyzing our previous microarray profiling. Expression of circRAD18 was found significantly upregulated in TNBC compared with normal mammary tissues and cell lines. circRAD18 was positively correlated with T stage, clinical stage and pathological grade and was an independent risk factor for TNBC patients. We performed proliferation, colony formation, cell migration, apoptosis and mouse xenograft assays to verify the functions of circRAD18. Knockdown of circRAD18 significantly suppressed cell proliferation and migration, promoted cell apoptosis and inhibited tumor growth in functional and xenograft experiments. Through luciferase reporter assays, we confirmed that circRAD18 acts as a sponge of miR-208a and miR-3164 and promotes TNBC progression through upregulating IGF1 and FGF2 expression. Altogether, our research revealed the pivotal role of circRAD18-miR-208a/3164-IGF1/FGF2 axis in TNBC tumorigenesis and metastasis though the mechanism of competing endogenous RNAs. Thus, circRAD18 may serve as a novel prognostic biomarker and potential target for TNBC treatment in the future.

Extracellular matrix protein anosmin-1 modulates olfactory ensheathing cell maturation in chick olfactory bulb development.

Olfactory ensheathing cells (OECs) are a specialized class of glia, wrapping around olfactory sensory axons that target the olfactory bulb (OB) and cross the peripheral nervous system/central nervous system boundary during development and continue to do so post-natally. OEC subpopulations perform distinct subtype-specific functions dependent on their maturity status. Disrupted OEC development is thought to be associated with abnormal OB morphogenesis, leading to anosmia, a defining characteristic of Kallmann syndrome. Hence, anosmin-1 encoded by Kallmann syndrome gene (KAL-1) might modulate OEC differentiation/maturation in the OB. We performed in ovo electroporation of shRNA in the olfactory placode to knock-down kal in chick embryos, resulting in abnormal OB morphogenesis and loss of olfactory sensory axonal innervation into OB. BLBP-expressing OECs appeared to form a thinner and poorly organized outmost OB layer where SOX10 expressing OECs were completely absent with emergence of GFAP-expressing OECs. Furthermore, in embryonic day 10 chick OB explant cultures, GFAP expression in OECs accumulating along the OB nerve layers was dramatically reduced by recombinant anosmin-1. We then purified immature OECs from embryonic day 10 chick OB. These cells express GFAP after 7 days in vitro, exhibiting a multipolar morphology. Overexpression of chick anosmin, exogenous anosmin-1 or FGF2 could inhibit GFAP expression with cells presenting elongated morphology, which was blocked by the FGF receptor inhibitor Su5402. These data demonstrate that anosmin-1 functions via FGF signalling in regulating OEC maturation, thereby providing a permissive glial environment for axonal innervation into the OB during development.

Proliferation of prostate epithelia induced by IL-6 from stroma reacted with Trichomonas vaginalis.

Benign prostatic hyperplasia (BPH) is characterized by the proliferation of stromal and epithelial cell types in the prostate, and interactions between the two types of cells. We demonstrated previously that proliferation of prostate stromal cells was induced by BPH epithelial cells in response to Trichomonas vaginalis (Tv) infection via crosstalk with mast cells. In this study, we investigated whether IL-6 released by the proliferating stromal cells in turn induce the BPH epithelial cells to multiply. When culture supernatants of the proliferating prostate stromal cells were added to BPH epithelial cells, the latter multiplied, and expression of cyclin D1, FGF2 and Bcl-2 increased. Blocking the IL-6 signalling pathway with anti-IL-6R antibody or JAK1/2 inhibitor inhibited the proliferation of the BPH epithelial cells and reduced the expression of IL-6, IL-6R and STAT3. Also, epithelial-mesenchymal transition was detected in the proliferating BPH epithelial cells. In conclusion, IL-6 released from proliferating prostate stromal cells induced by BPH epithelial cells infected with Tv in turn induces multiplication of the BPH epithelial cells. This result provides first evidence that the inflammatory microenvironment of prostate stromal cells resulting from Tv infection induces the proliferation of prostate epithelial cells by stromal-epithelial interaction.

Prognostic importance of FGF2 and FGFR1 expression for patients affected by ameloblastoma.

BACKGROUND: Fibroblast growth factor 2 (FGF2) and FGF receptor 1 (FGFR1) have been investigated in different human neoplasms and were shown to play important roles in the pathogenesis of these diseases; however, very few are known regarding their prognostic importance in the context of ameloblastoma. Therefore, the aim of this study was to investigate whether the expression of FGF2 and FGFR1 is associated with ameloblastoma clinical behavior. METHODS: Fifty-eight cases of ameloblastoma arranged in tissue microarray were submitted to immunohistochemistry against FGF2 and FGFR1. Clinicopathological parameters regarding sex, age, tumor size, duration and location, treatment, recurrences, radiographic features, cortical disruptions, and follow-up data were obtained from patients' medical records and correlated with the molecules expression. Univariate and multivariate Cox regression analyses were used to investigate the prognostic potential of the biomarkers. RESULTS: Forty-four cases (75.9%) exhibited cytoplasmic positivity for FGF2 in central and peripheral epithelial cells, 46 of 58 (79.3%) showed FGFR1 cytoplasmic positivity predominantly in the columnar peripheral cells, and 43 cases (74.1%) were positive for both. Expression of FGF2 and FGF2 + FGFR1 was associated with tumor recurrences (P = .05). However, univariate and multivariate analyses did not demonstrate a significant influence of FGF2, FGFR1, or FGF2 + FGFR1 in the 5-year disease-free survival (DFS) rate (P = .27, P = .33, and P = .25, respectively). CONCLUSION: Cytoplasmic expression of FGF2 and FGF2 + FGFR1 is associated with ameloblastoma recurrence, but FGF2 and FGFR1 are not determinants of a lower DFS.

[The Novel Short Isoform of Securin Stimulates the Expression of Cyclin D3 and Angiogenesis Factors VEGFA and FGF2, but Does Not Affect the Expression of MYC Transcription Factor].

Pituitary tumor-transforming gene-1 (PTTG1) encodes securin, a multifunctional protein involved in development of various types of cancer. Securin participates in the regulation of sister chromatids separation and the expression of multiple genes involved in the control of the cell cycle, metabolism, and angiogenesis. In several human cell lines, we have found a novel short isoform of securin mRNA, which does not contain exons 3 and 4. After the translation of this new mRNA, a shortened protein is produced that, like the full-size form, is able to activate the transcription of cyclin D3 gene (CCND3), which controls the G1/S transition and angiogenesis factors VEGFA (vascular endothelial growth factor), and FGF2 (fibroblast growth factor 2) in HEK293 cells. However, unlike the full-size protein, the short isoform of PTTG1 does not affect the MYC gene expression because it lacks the DNA-binding domain, which is needed for its interactions with the MYC promoter. Furthermore, the short form of securin does not influence the expression of MYC transcriptional targets, such as TP53 and IL-8. Thus, we found a novel isoform of securin which is able to activate a more restricted repertoire of genes compared to the full-size protein.

Optogenetic Inhibition of Striatal GABAergic Neuronal Activity Improves Outcomes After Ischemic Brain Injury.

BACKGROUND AND PURPOSE: Striatal GABAergic neuron is known as a key regulator in adult neurogenesis. However, the specific role of striatal GABAergic neuronal activity in the promotion of neurological recovery after ischemic stroke remains unknown. Here, we used optogenetic approach to investigate these effects and mechanism. METHODS: Laser stimulation was delivered via an implanted optical fiber to inhibit or activate the striatal GABAergic neurons in Gad2-Arch-GFP or Gad2-ChR2-tdTomato mice (n=80) 1 week after 60-minute transient middle cerebral artery occlusion. Neurological severity score, brain atrophy volume, microvessel density, and cell morphological changes were examined using immunohistochemistry. Gene expression and protein levels of related growth factors were further examined using real-time polymerase chain reaction and Western blotting. RESULTS: Inhibiting striatal GABAergic neuronal activity improved functional recovery, reduced brain atrophy volume, and prohibited cell death compared with the control (P<0.05). Microvessel density and bFGF (basic fibroblast growth factor) expression in the inhibition group were also increased (P<0.05). In contrast, activation of striatal GABAergic neurons resulted in adverse effects compared with the control (P<0.05). Using cocultures of GABAergic neurons, astrocytes, and endothelial cells, we further demonstrated that the photoinhibition of GABAergic neuronal activity could upregulate bFGF expression in endothelial cells, depending on the presence of astrocytes. The conditioned medium from the aforementioned photoinhibited 3-cell coculture system protected cells from oxygen glucose deprivation injury. CONCLUSIONS: After ischemic stroke, optogenetic inhibition of GABAergic neurons upregulated bFGF expression by endothelial cells and promoted neurobehavioral recovery, possibly orchestrated by astrocytes. Optogenetically inhibiting neuronal activity provides a novel approach to promote neurological recovery.

Suppression of Basic Fibroblast Growth Factor Expression by Antisense Oligonucleotides Inhibits Neural Stem Cell Proliferation and Differentiation in Rat models With Focal Cerebral Infarction.

This study is designed to investigate the role of basic fibroblast growth factor (bFGF) antisense oligonucleotide (ASODN) on the proliferation and differentiation of neural stem cells (NSCs) in rat models with focal cerebral infarction (CI). Seventy-five Sprague-Dawlay (SD) rats were randomly divided into the control, sham, middle cerebral artery occlusion (MCAO), MCAO + nonsense oligonucleotide (NODN), and MCAO + ASODN groups. Proliferation and differentiation of NSCs were detected by bromodeoxyuridine (BrdU) and immunofluorescence staining, respectively. ELISA was performed to detect the expressions of endogenous factors that include insulin-like growth factor 1 (IGF-1), glial cell line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), transforming growth factor-α1 (TGF-α1), bFGF, and nerve growth factor (NGF). Results show significant neurological deficits and focal CI in the MCAO and MCAO + NODN groups. An obvious increase of NSC proliferation, reactive proliferation of astrocytes in CI areas, differentiation of newly proliferated NSCs into mature neuronal cells, and expressions of endogenous growth factors exhibited in the MCAO, MCAO + NODN and MCAO + ASODN groups. Compared to the MCAO and MACO + NODN groups, the MCAO + ASODN group showed a significant decrease NSC proliferation and differentiation in CI areas as well as decrease expressions of endogenous growth factors. These findings may offer insight to help us understand more as to how bFGF ASODN can effectively suppress the proliferation and differentiation of NSCs. These findings are expected to help contribute to research for new targets in the treatment of focal CI. J. Cell. Biochem. 118: 3875-3882, 2017. © 2017 Wiley Periodicals, Inc.

Role of Daptomycin on Burn Wound Healing in an Animal Methicillin-Resistant Staphylococcus aureus Infection Model.

Prolonged hospitalization and antibiotic therapy are risk factors for the development of methicillin-resistant Staphylococcus aureus (MRSA) infections in thermal burn patients. We used a rat model to study the in vivo efficacy of daptomycin in the treatment of burn wound infections by S. aureus, and we evaluated the wound healing process through morphological and immunohistochemical analysis. A copper bar heated in boiling water was applied on a paraspinal site of each rat, resulting in two full-thickness burns. A small gauze was placed over each burn and inoculated with 5 × 107 CFU of S. aureus ATCC 43300. The study included two uninfected control groups with and without daptomycin treatment, an infected control group that did not receive any treatment, and two infected groups treated, respectively, with intraperitoneal daptomycin and teicoplanin. The main outcome measures were quantitative culture, histological evaluation of tissue repair, and immunohistochemical expression of wound healing markers: epidermal growth factor receptor (EGFR) and fibroblast growth factor 2 (FGF-2). The highest inhibition of infection was achieved in the group that received daptomycin, which reduced the bacterial load from 107 CFU/ml to about 103 CFU/g (P < 0.01). The groups treated with daptomycin showed better overall healing with epithelialization and significantly higher collagen scores than the other groups, and these findings were also confirmed by immunohistochemical data. In conclusion, our results support the hypothesis that daptomycin is an important modulator of wound repair by possibly reducing hypertrophic burn scar formation.

MicroRNA-29a in Adult Muscle Stem Cells Controls Skeletal Muscle Regeneration During Injury and Exercise Downstream of Fibroblast Growth Factor-2.

The expansion of myogenic progenitors (MPs) in the adult muscle stem cell niche is critical for the regeneration of skeletal muscle. Activation of quiescent MPs depends on the dismantling of the basement membrane and increased access to growth factors such as fibroblast growth factor-2 (FGF2). Here, we demonstrate using microRNA (miRNA) profiling in mouse and human myoblasts that the capacity of FGF2 to stimulate myoblast proliferation is mediated by miR-29a. FGF2 induces miR-29a expression and inhibition of miR-29a using pharmacological or genetic deletion decreases myoblast proliferation. Next generation RNA sequencing from miR-29a knockout myoblasts (Pax7(CE/+) ; miR-29a(flox/flox) ) identified members of the basement membrane as the most abundant miR-29a targets. Using gain- and loss-of-function experiments, we confirm that miR-29a coordinately regulates Fbn1, Lamc1, Nid2, Col4a1, Hspg2 and Sparc in myoblasts in vitro and in MPs in vivo. Induction of FGF2 and miR-29a and downregulation of its target genes precedes muscle regeneration during cardiotoxin (CTX)-induced muscle injury. Importantly, MP-specific tamoxifen-induced deletion of miR-29a in adult skeletal muscle decreased the proliferation and formation of newly formed myofibers during both CTX-induced muscle injury and after a single bout of eccentric exercise. Our results identify a novel miRNA-based checkpoint of the basement membrane in the adult muscle stem cell niche. Strategies targeting miR-29a might provide useful clinical approaches to maintain muscle mass in disease states such as ageing that involve aberrant FGF2 signaling.

A Primitive Growth Factor, NME7AB , Is Sufficient to Induce Stable Naïve State Human Pluripotency; Reprogramming in This Novel Growth Factor Confers Superior Differentiation.

Scientists have generated human stem cells that in some respects mimic mouse naïve cells, but their dependence on the addition of several extrinsic agents, and their propensity to develop abnormal karyotype calls into question their resemblance to a naturally occurring "naïve" state in humans. Here, we report that a recombinant, truncated human NME7, referred to as NME7AB here, induces a stable naïve-like state in human embryonic stem cells and induced pluripotent stem cells without the use of inhibitors, transgenes, leukemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), feeder cells, or their conditioned media. Evidence of a naïve state includes reactivation of the second X chromosome in female source cells, increased expression of naïve markers and decreased expression of primed state markers, ability to be clonally expanded and increased differentiation potential. RNA-seq analysis shows vast differences between the parent FGF2 grown, primed state cells, and NME7AB converted cells, but similarities to altered gene expression patterns reported by others generating naïve-like stem cells via the use of biochemical inhibitors. Experiments presented here, in combination with our previous work, suggest a mechanistic model of how human stem cells regulate self-replication: an early naïve state driven by NME7, which cannot itself limit self-replication and a later naïve state regulated by NME1, which limits self-replication when its multimerization state shifts from the active dimer to the inactive hexamer.

Dual Functional MicroRNA-186-5p Targets both FGF2 and RelA to Suppress Tumorigenesis of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is one of the most malignant cancers. MicroRNAs (miRs) were reported to play important roles in GBM recently. However, the role of a novel miR-186-5p in GBM tumorigenesis is still elusive. Using bioinformatics, miR-186-5p was identified as potential regulators of both fibroblast growth factor (FGF)-2 and NF-κB subunit RelA. Luciferase reporter assay was used to confirm the direct recognition FGF2 and RelA mRNAs by miR-186-5p. Invasion and migration assays were employed to study the effect of miR-186-5p on GBM cell growth in vitro. Xenograft tumor animal model was established to elucidate the in vivo function of miR-186-5p. MiR-186-5p directly targeted mRNAs of both FGF2 and RelA, and repressed their expressions. Invasive and migratory abilities of GBM cells and growth of xenograft tumors were significantly inhibited by miR-186-5p, which can be restored by re-introduction of FGF2 and RelA expressions. MiR-186-5p is a novel tumor suppressor miR that functions to inhibit tumorigenesis of GBM both in vitro and in vivo, by targeting both FGF2 and RelA. MiR-186-5p/FGF2/RelA pathway may be potentially used as molecular targets of in the clinical treatment of GBM.

Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells.

Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H6) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN6) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H6-FGF2, FGF2-H6, HN6-FGF2, and FGF2-HN6) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H6-FGF2, FGF2-H6, HN6-FGF2, and FGF2-HN6 were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility.

Impact of Rantes from jawbone on Chronic Fatigue Syndrome.

This study elucidates the question of whether chronic inflammation in the jawbone contributes to the development of Chronic Fatigue Syndrome (CFS). Fatty degenerative osteonecrosis in jawbone (FDOJ) may contribute to CFS by induction of inflammatory mediators. We examined seven cytokines by multiplex analysis in jawbone samples from two groups of patients. In order to clarify neurological interrelations, specimens from 21 CFS patients were analyzed from areas of previous surgery in the retromolar wisdom tooth area. Each of the retromolar jawbone samples showed clinically fatty degenerated and osteonecrotic medullary changes. As control, healthy jawbone specimens from 19 healthy patients were analyzed. All fatty necrotic and osteolytic jawbone (FDOJ) samples showed high expression of RANTES and fibroblast growth factor (FGF)-2. FDOJ cohorts showed a 30-fold mean overexpression of RANTES and a 20-fold overexpressed level of FGF-2 when compared to healthy controls. As RANTES is discussed in the literature as a possible contributor to inflammatory diseases, we hypothesize that FDOJ in areas of improper and incomplete wound healing in the jawbone may hyperactivate signaling pathways. Constituting a hidden source of “silent inflammation” FDOJ may represent a hitherto unknown cause for the development of CFS.

Therapeutic Benefits and Adverse Effects of Combined Proangiogenic Gene Therapy in Mouse Critical Leg Ischemia.

BACKGROUND: Critical leg ischemia (CLI) represents the ultimate stage of peripheral arterial disease. Despite current surgery advances, patients with CLI have limited therapeutic options. Therapeutic angiogenesis thus appears as a powerful approach, aiming to stimulate vessel formation by angiogenic molecules administration. In this context, combined gene therapy has been proved to be the most efficient. The present study aims to compare, in a preclinical mouse model, the therapeutic benefit of a combination of 2 angiogenic factors fibroblast growth factor 2 (FGF2) and Cyr61 using plasmid and viral vectors, able to generate short- or long-term transgene expression in the leg, respectively. METHODS: Two therapeutic genes, FGF2 and Cyr61, were introduced into internal ribosome entry site-based expression vectors (FGFiCyr) allowing co-expression of the 2 transgenes. The proangiogenic plasmid pC-FGFiCyr was assessed by intramuscular administration followed by electrotransfer into ischemic legs. To generate long-term transgene expression, the FGFiCyr bicistronic cassette was introduced into an adenoassociated virus-derived vector (rAAV). The rAAV treatment was performed either before or immediately after surgery. Therapeutic effects were analyzed by laser Doppler imaging, clinical score, and angiography. RESULTS: The plasmid pC-FGFiCyr improved revascularization, reperfusion, and clinical score. Surprisingly, when AAV-FGFiCyr was injected 21 or 28 days before surgery, the proangiogenic rAAV was drastically deleterious on all measured parameters. In contrast, when administrated shortly after surgery, AAV-FGFiCyr generated therapeutic benefits, with a significantly better clinical score than after treatment with the plasmid. CONCLUSIONS: Therapeutic effects of the angiogenic combination FGF2-Cyr61 is observed with short-term transgene expression, but the treatment is significantly more efficient when a long-term expression viral vector is used. However, the rAAV-FGFiCyr generated therapeutic benefit only when injected in an ischemic leg, whereas the same dose of rAAV exhibited deleterious effects when administrated to healthy animals. These data may contribute to the understanding of the moderate success of proangiogenic treatments in CLI gene therapy clinical assays.

Opposing effects of Sca-1(+) cell-based systemic FGF2 gene transfer strategy on lumbar versus caudal vertebrae in the mouse.

Our previous work showed that a Sca-1(+) cell-based FGF2 therapy was capable of promoting robust increases in trabecular bone formation and connectivity on the endosteum of long bones. Past work reported that administration of FGF2 protein promoted bone formation in red marrow but not in yellow marrow. The issue as to whether the Sca-1(+) cell-based FGF2 therapy is effective in yellow marrow is highly relevant to its clinical potential for osteoporosis, as most red marrows in a person of an advanced age are converted to yellow marrows. Accordingly, this study sought to compare the osteogenic effects of this stem cell-based FGF2 therapy on red marrow-filled lumbar vertebrae with those on yellow marrow-filled caudal vertebrae of young adult W(41)/W(41) mice. The Sca-1(+) cell-based FGF2 therapy drastically increased trabecular bone formation in lumbar vertebrae, but the therapy not only did not promote bone formation but instead caused substantial loss of trabecular bone in caudal vertebrae. The lack of an osteogenic response was not due to insufficient engraftment of FGF2-expressing Sca-1(+) cells or inadequate FGF2 expression in caudal vertebrae. Previous studies have demonstrated that recipient mice of this stem cell-based FGF2 therapy developed secondary hyperparathyroidism and increased bone resorption. Thus, the loss of bone mass in caudal vertebrae might in part be due to an increase in resorption without a corresponding increase in bone formation. In conclusion, the Sca-1(+) cell-based FGF2 therapy is osteogenic in red marrow but not in yellow marrow.