Unconventional secretion mediated by direct protein self-translocation across the plasma membranes of mammalian cells.

In recent years, a surprisingly complex picture emerged about endoplasmic reticulum (ER)/Golgi-independent secretory pathways, and several routes have been discovered that differ with regard to their molecular mechanisms and machineries. Fibroblast growth factor 2 (FGF2) is secreted by a pathway of unconventional protein secretion (UPS) that is based on direct self-translocation across the plasma membrane. Building on previous research, a component of this process has been identified to be glypican-1 (GPC1), a GPI-anchored heparan sulfate proteoglycan located on cell surfaces. These findings not only shed light on the molecular mechanism underlying this process but also reveal an intimate relationship between FGF2 and GPC1 that might be of critical relevance for the prominent roles they both have in tumor progression and metastasis.

Fibroblast growth factor 2 enhances BMSC stemness through ITGA2-dependent PI3K/AKT pathway activation.

Bone marrow-derived mesenchymal stem cells (BMSC) are promising cellular reservoirs for treating degenerative diseases, tissue injuries, and immune system disorders. However, the stemness of BMSCs tends to decrease during in vitro cultivation, thereby restricting their efficacy in clinical applications. Consequently, investigating strategies that bolster the preservation of BMSC stemness and maximize therapeutic potential is necessary. Transcriptomic and single-cell sequencing methodologies were used to perform a comprehensive examination of BMSCs with the objective of substantiating the pivotal involvement of fibroblast growth factor 2 (FGF2) and integrin alpha 2 (ITGA2) in stemness regulation. To investigate the impact of these genes on the BMSC stemness in vitro, experimental approaches involving loss and gain of function were implemented. These approaches encompassed the modulation of FGF2 and ITGA2 expression levels via small interfering RNA and overexpression plasmids. Furthermore, we examined their influence on the proliferation and differentiation capacities of BMSCs, along with the expression of stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, and sex determining region Y-box 2. Transcriptomic analyzes successfully identified FGF2 and ITGA2 as pivotal genes responsible for regulating the stemness of BMSCs. Subsequent single-cell sequencing revealed that elevated FGF2 and ITGA2 expression levels within specific stem cell subpopulations are closely associated with stemness maintenance. Moreover, additional in vitro experiments have convincingly demonstrated that FGF2 effectively enhances the BMSC stemness by upregulating ITGA2 expression, a process mediated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. This conclusion was supported by the observed upregulation of stemness markers following the induction of FGF2 and ITGA2. Moreover, administration of the BEZ235 pathway inhibitor resulted in the repression of stemness transcription factors, suggesting the substantial involvement of the PI3K/AKT pathway in stemness preservation facilitated by FGF2 and ITGA2. This study elucidates the involvement of FGF2 in augmenting BMSC stemness by modulating ITGA2 and activating the PI3K/AKT pathway. These findings offer valuable contributions to stem cell biology and emphasize the potential of manipulating FGF2 and ITGA2 to optimize BMSCs for therapeutic purposes.

Piezo1 (Piezo-Type Mechanosensitive Ion Channel Component 1)-Mediated Mechanosensation in Macrophages Impairs Perfusion Recovery After Hindlimb Ischemia in Mice.

BACKGROUND: Angiogenesis is a promising strategy for those with peripheral artery disease. Macrophage-centered inflammation is intended to govern the deficiency of the angiogenic response after hindlimb ischemia. However, little is known about the mechanism of macrophage activation beyond signals from cytokines and chemokines. We sought to identify a novel mechanical signal from the ischemic microenvironment that provokes macrophages and the subsequent inflammatory cascade and to investigate the potential role of Piezo-type mechanosensitive ion channels (Piezo) on macrophages during this process. METHODS: Myeloid cell-specific Piezo1 (Piezo-type mechanosensitive ion channel component 1) knockout (Piezo1ΔMΦ) mice were generated by crossing Piezo1fl/fl (LysM-Cre-/-; Piezo1 flox/flox) mice with LysM-Cre transgenic mice to assess the roles of Piezo1 in macrophages after hindlimb ischemia. Furthermore, in vitro studies were carried out in bone marrow-derived macrophages to decipher the underlying mechanism. RESULTS: We found that tissue stiffness gradually increased after hindlimb ischemia, as indicated by Young's modulus. Compared to Piezo2, Piezo1 expression and activation were markedly upregulated in macrophages from ischemic tissues in concurrence with increased tissue stiffness. Piezo1ΔMΦ mice exhibited improved perfusion recovery by enhancing angiogenesis. Matrigel tube formation assays revealed that Piezo1 deletion promoted angiogenesis by enhancing FGF2 (fibroblast growth factor-2) paracrine signaling in macrophages. Conversely, activation of Piezo1 by increased stiffness or the agonist Yoda1 led to reduced FGF2 production in bone marrow-derived macrophages, which could be blocked by Piezo1 silencing. Mechanistically, Piezo1 mediated extracellular Ca2+ influx and activated Ca2+-dependent CaMKII (calcium/calmodulin-dependent protein kinase II)/ETS1 (ETS proto-oncogene 1) signaling, leading to transcriptional inactivation of FGF2. CONCLUSIONS: This study uncovers a crucial role of microenvironmental stiffness in exacerbating the macrophage-dependent deficient angiogenic response. Deletion of macrophage Piezo1 promotes perfusion recovery after hindlimb ischemia through CaMKII/ETS1-mediated transcriptional activation of FGF2. This provides a promising therapeutic strategy to enhance angiogenesis in ischemic diseases.

Serum fibroblast growth factor-2 levels complement vital biomarkers for diagnosing heart failure.

BACKGROUND: Early diagnosis of atrial fibrillation is important as it is crucial for improving patient outcomes. Fibroblast growth factor-2 (FGF2) may serve as a diagnostic biomarker for heart failure due to its ability to promote cardiac fibrosis and hypertrophy; however, the relationship between FGF2 concentration and heart failure is unclear. Therefore, this study aimed to explore whether FGF2 could aid in distinguishing patients with heart failure from healthy controls and those with dyspnea without heart failure. Additionally, to evaluate the possible correlation between serum FGF2 levels and its diagnostic parameters in patients with heart failure. METHODS: Plasma FGF2 concentration was measured in 114 patients with a complaint of dyspnea (enrolled in the study between January 2022 and August 2022). Based on heart failure diagnosis, the patients were assigned to three groups, as follows: heart failure (n = 80), non-heart-failure dyspnea (n = 34), and healthy controls (n = 36), following physical examination. Possible correlations between serum FGF2 levels and other prognostic parameters in patients with heart failure were analyzed. RESULTS: Serum FGF2 levels were higher in patients with heart failure (125.60 [88.95, 183.40] pg/mL) than in those with non-heart-failure dyspnea (65.30 [28.85, 78.95] pg/mL) and healthy controls (78.90 [60.80, 87.20] pg/mL) (p < 0.001). Receiver operating characteristic curve analysis identified FGF2 concentration as a significant predictor in heart failure diagnosis, with an area under the curve of 0.8693 (p < 0.0001). Importantly, in the heart failure group, serum FGF2 concentrations correlated with key prognostic parameters for heart failure, such as reduced left ventricular ejection fraction and elevated serum levels of N-terminal pro-B-type natriuretic peptide. CONCLUSIONS: Elevated serum FGF2 level is strongly associated with an increased risk of heart failure and could serve as a useful biomarker to complement vital diagnostic parameters for heart failure.

Extracellular matrix hydrogels with fibroblast growth factor 2 containing exosomes for reconstructing skin microstructures.

Decellularized extracellular matrix hydrogel (ECM hydrogel), a natural material derived from normal tissue with unique biocompatibility properties, is widely used for tissue repair. However, there are still problems such as poor biological activity and insufficient antimicrobial property. To overcome these drawbacks, fibroblast growth factor 2 (FGF 2) containing exosome (exoFGF 2) was prepared to increase the biological activity. Furthermore, the antimicrobial capacity of ECM hydrogel was optimised by using copper ions as a ligand-bonded cross-linking agent. The decellularized extracellular matrix hydrogel, intricately cross-linked with copper ions through ligand bonds and loaded with FGF 2 containing exosome (exoFGF 2@ECM/Cu2+ hydrogel), has demonstrated exceptional biocompatibility and antimicrobial properties. In vitro, exoFGF 2@ECM/Cu2+ hydrogel effectively promoted cell proliferation, migration, antioxidant and inhibited bacterial growth. In vivo, the wound area of rat treated with exoFGF 2@ECM/Cu2+ hydrogels were significantly smaller than that of other groups at Day 5 (45.24% ± 3.15%), Day 10 (92.20% ± 2.31%) and Day 15 (95.22% ± 1.28%). Histological examination showed that exoFGF 2@ECM/Cu2+ hydrogels promoted angiogenesis and collagen deposition. Overall, this hydrogel has the potential to inhibit bacterial growth and effectively promote wound healing in a variety of clinical applications.

Long Non-coding RNA LINC00342 Promotes the Proliferation, Invasion, and Migration of Primary Hepatocellular Carcinoma Cells by Regulating the Expression of miRNA-19a-3p, miRNA-545-5p, and miRNA-203a-3p.

This study aimed to investigate the role of the long non-coding RNA (lncRNA) LINC00342-207 (LINC00342) in the development and progression of primary hepatocellular carcinoma (HCC). Forty-two surgically resected HCC tissues and corresponding paracancerous tissues were collected from October 2019 to December 2020 and examined for lncRNA LINC00342, microRNA (miR)-19a-3p, miR-545-5p, miR-203a-3p, cell cycle protein D1 (CyclinD1/CCND1), murine double minute 2 (MDM2), and fibroblast growth factor 2 (FGF2) expression. The disease-free survival and overall survival of patients with HCC were followed up. HCC cell lines and the normal hepatocyte cell line HL-7702 were cultured and the expression level of LINC00342 was measured. HepG2 cells were transfected with LINC00342 siRNA, LINC00342 overexpression plasmid, miR-19a-3p mimics and their corresponding suppressors, miR-545-5p mimics and their corresponding suppressors, and miR-203a-3p mimics and their corresponding suppressors. The proliferation, apoptosis, migration, and invasion of HepG2 cells were detected. Stably transfected HepG2 cells were inoculated into the left axilla of male BALB/c nude mice, and the volume and quality of transplanted tumors as well as the expression levels of LINC00342, miR-19a-3p, miR-545-5p, miR-203a-3p, CCND1, MDM2, and FGF2 were examined. LINC00342 played an oncogenic role in HCC and exhibited inhibitory effects on proliferation, migration, and invasion, and promoted the apoptosis of HepG2 cells. Moreover, it inhibited the growth of transplanted tumors in vivo in mice. Mechanistically, the oncogenic effect of LINC00342 was associated with the targeted regulation of the miR-19a-3p/CCND1, miR-545-5p/MDM2, and miR-203a-3p/FGF2 axes.

Periodontal regenerative therapy using recombinant human fibroblast growth factor (rhFGF)-2 in combination with carbonate apatite granules or rhFGF-2 alone: 12-month randomized controlled trial.

OBJECTIVES: This randomized controlled trial compared the outcomes of recombinant human fibroblast growth factor (rhFGF)-2 plus carbonate apatite (CO3Ap) granules with rhFGF-2 alone in the treatment of intrabony periodontal defects. MATERIALS AND METHODS: Patients with Stage III Grade B/C periodontitis who had completed initial periodontal therapy and had intrabony defects with a depth of ≥ 3 mm were included. Defects were treated solely with rhFGF-2 (control) or rhFGF-2 plus CO3Ap (test). Periodontal parameters and a patient-reported outcome measure (PROM) were assessed at baseline, at 6, 9 and 12 months postoperatively. The primary outcome was the change in clinical attachment level (CAL) from baseline to 12 months postoperatively. Using the Friedman test with Dunn's post-test, intragroup data were compared over time, and Mann-Whitney U test was used to assess intergroup data at each time point. RESULTS: Forty-eight sites in 38 patients were subjected to analysis. At 12 months postoperatively, CAL in both groups showed a significant improvement from baseline (p < 0.001). CAL gain was 3.4 ± 1.3 mm in the test group and 3.2 ± 1.2 mm in the control group, with no significant intergroup difference (p = 0.567). Radiographic bone fill in the test group (67.2%) was significantly greater than in the control group (32.4%) (p < 0.001). PROM scores showed no difference between groups. CONCLUSIONS: At 12 months, the outcomes including CAL gain and PROM showed no significant differences between groups, although the combination treatment enhanced radiographic bone fill. CLINICAL RELEVANCE: The use of rhFGF-2 (with/without CO3Ap) could lead to significant improvement in clinical parameters in the treatment of intrabony periodontal defects. The benefit of adding CO3Ap to rhFGF-2 therapy needs further evaluation. CLINICAL TRIAL REGISTRATION NUMBER: The University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) : UMIN000040783.

Lack of Direct Effects of Neurotrophic Factors in an In Vitro Model of Neuroinflammation.

Neuroinflammation is associated with several neurological disorders including temporal lobe epilepsy. Seizures themselves can induce neuroinflammation. In an in vivo model of epilepsy, the supplementation of brain-derived neurotropic factor (BDNF) and fibroblast growth factor-2 (FGF-2) using a Herpes-based vector reduced epileptogenesis-associated neuroinflammation. The aim of this study was to test whether the attenuation of the neuroinflammation obtained in vivo with BDNF and FGF-2 was direct or secondary to other effects, for example, the reduction in the severity and frequency of spontaneous recurrent seizures. An in vitro model of neuroinflammation induced by lipopolysaccharide (LPS, 100 ng/mL) in a mouse primary mixed glial culture was used. The releases of cytokines and NO were analyzed via ELISA and Griess assay, respectively. The effects of LPS and neurotrophic factors on cell viability were determined by performing an MTT assay. BDNF and FGF-2 were tested alone and co-administered. LPS induced a significant increase in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and NO. BDNF, FGF-2, and their co-administration did not counteract these LPS effects. Our study suggests that the anti-inflammatory effect of BDNF and FGF-2 in vivo in the epilepsy model was indirect and likely due to a reduction in seizure frequency and severity.

Fibroblast Growth Factor 2 Is Produced By Renal Tubular Cells to Act as a Paracrine Factor in Maladaptive Kidney Repair After Cisplatin Nephrotoxicity.

Kidney repair after injury involves the cross-talk of injured kidney tubules with interstitial fibroblasts and immune cells. Although tubular cells produce multiple cytokines, the role and regulation of specific cytokines in kidney repair are largely undefined. In this study, we detected the induction of fibroblast growth factor 2 (FGF2) in mouse kidneys after repeated low-dose cisplatin (RLDC) treatment and in RLDC-treated renal proximal tubule cells in vitro. We further detected FGF2 in the culture medium of RLDC-treated renal tubular cells but not in the medium of control cells, indicating that RLDC induces FGF2 expression and secretion. Compared with the medium of control cells, the medium of RLDC-treated renal tubular cells was twice as effective in promoting fibroblast proliferation. Remarkably, the proliferative effect of the RLDC-treated cell medium was diminished by FGF2-neutralizing antibodies. In addition, the RLDC-treated cell medium induced the expression of fibrosis-related proteins, which was partially suppressed by FGF2-neutralizing antibodies. In mice, FGF2 deficiency partially prevented RLDC-induced decline in kidney function, loss of kidney weight, renal fibrosis, and inflammation. Together, these results indicate that FGF2 is produced by renal tubular cells after kidney injury and acts as an important paracrine factor in maladaptive kidney repair and disease progression.

MIR-107/HMGB1/FGF-2 axis responds to excessive mechanical stretch to promote rapid repair of vascular endothelial cells.

The increase of vascular wall tension can lead to endothelial injury during hypertension, but its potential mechanism remains to be studied. Our results of previous study showed that HUVECs could induce changes in HMGB1/RAGE to resist abnormal mechanical environments in pathological mechanical stretching. In this study, we applied two different kinds of mechanical tension to endothelial cells using the in vitro mechanical loading system FlexCell-5000T and focused on exploring the expression of miR-107 related pathways in HUVECs with excessive mechanical tension. The results showed that miR-107 negatively regulated the expression of the HMGB1/RAGE axis under excessive mechanical tension. Excessive mechanical stretching reduced the expression of miR-107 in HUVECs, and increased the expression of the HMGB1/RAGE axis. When miR-107 analog was transfected into HUVECs with lipo3000 reagent, the overexpression of miR-107 slowed down the increase of the HMGB1/RAGE axis caused by excessive mechanical stretching. At the same time, the overexpression of miR-107 inhibited the proliferation and migration of HUVECs to a certain extent. On the contrary, when miR-107 was silent, the proliferation and migration of HUVECs showed an upward trend. In addition, the study also showed that under excessive mechanical tension, miR-107 could regulate the expression of FGF-2 by HMGB1. In conclusion, these findings suggest that pathological mechanical stretching promote resistance to abnormal mechanical stimulation on HUVECs through miR-107/HMGB1/RAGE/FGF-2 pathway, thus promote vascular repair after endothelial injury. The suggest that miR-107 is a potential therapeutic target for hypertension.

The BMP and FGF pathways reciprocally regulate odontoblast differentiation.

PURPOSE: Previous studies demonstrated that the exposure of primary dental pulp (DP) cultures to fibroblast growth factor 2 (FGF2) between days 3-7 exerted significant and long-lasting stimulatory effects on odontoblast differentiation and Dspp expression. These effects involved the increased expression of components of bone morphogenetic protein (BMP) signaling and were reverted by a BMP inhibitor noggin. FGF2 also transiently stimulated osteoblast differentiation and the expression of Ibsp and Dmp1. The present study aimed to further explore interactions between BMP and FGF signaling during odontoblast and osteoblast differentiation in DP cultures. MATERIALS AND METHODS: Cultures were established using DP tissue isolated from non-transgenic and fluorescent reporter (DSPP-Cerulean, BSP-GFP, and DMP1-mCherry) transgenic mice and exposed to BMP2, FGF2, SU5402 (an FGF receptor inhibitor), and noggin between days 3-7. Mineralization, gene expression, fluorescent protein expression, and odontoblast formation were examined using xylenol orange, quantitative PCR, fluorometric analysis, and immunocytochemistry, respectively. RESULTS: BMP2 activated SMAD1/5/8 but not ERK1/2 signaling, whereas FGF2 exerted opposite effects. BMP2 did not affect mineralization, the expression of Ibsp and Dmp1, and the percentage of DSPP-Cerulean+ odontoblasts but significantly increased Dspp and DSPP-Cerulean. In cultures exposed to BMP2 and FGF2, respectively, both SU5402 and noggin led to long-lasting decreases in Dspp and DSPP-Cerulean and transient decreases in Dmp1 and DMP1-mCherry without affecting Ibsp and BSP-GFP. CONCLUSION: BMP2 and FGF2 exerted reciprocal stimulatory effects on odontoblast differentiation, whereas their effects on osteoblast differentiation were mediated independently. These data will further elucidate the perspectives of using BMP2 and FGF2 for dentin regeneration/repair.

Immunovascular microenvironment in relation to prognostic heterogeneity of WNT/ß-catenin-activated hepatocellular carcinoma.

AIM: WNT/ß-catenin-activated hepatocellular carcinoma (W/B subclass HCC) is considered a molecularly homogeneous entity and has been linked to resistance to immunotherapy. However, recent studies have indicated possible heterogeneity in the immunovascular microenvironment in this subclass. We set out to test the hypothesis that specific immunovascular features might stratify W/B subclass HCCs into tumors having distinct aggressive natures. METHODS: In this study, we analyzed 352 resected HCCs including 78 immunohistochemically defined W/B subclass HCCs. The density of tumor-infiltrating CD3+ T cells and the area ratio of vessels encapsulating tumor clusters (VETC) were calculated on tissue specimens. The gene expressions of angiogenic factors were measured by quantitative reverse transcription-polymerase chain reaction. Disease-free survival (DFS) was assessed using multivariable Cox regression analyses. RESULTS: The T-cell density of W/B subclass HCCs was regionally heterogenous within tumor tissues, and focally reduced T-cell density was observed in areas with VETC. VETC-positivity (defined as VETC area ratio greater than 1%) was inversely associated with T-cell infiltration in both W/B subclass and non-W/B subclass HCCs. Fibroblast growth factor 2 (FGF2) gene expression was higher in W/B subclass than in non-W/B subclass HCCs. The VETC-positivity and low T-cell density correlated with increased expression of FGF2 in W/B subclass HCCs. Additionally, VETC-positive HCCs showed significantly shorter DFS in W/B subclass HCCs. CONCLUSIONS: In conclusion, the immune and vascular microenvironments are interrelated and are also correlated with clinicopathological heterogeneity in W/B subclass HCC. These results could inform clinical practice and translational research on the development of therapeutic stratification of HCCs.

Safety of terminally gamma-ray-sterilized screws coated with fibroblast growth factor 2-calcium phosphate composite layers in non-human primates.

Screws coated with fibroblast growth factor 2 (FGF-2)-calcium phosphate (CP) composite layers exhibit enhanced soft tissue and bone formation and angiogenesis because of the biological activity of FGF-2. Furthermore, the mitogenic activity of the FGF-2 within the composite layers remains unchanged after gamma-ray sterilization, which may improve the storage stability prior to clinical use. However, the in vivo safeties of these screws as spinal implants remain unknown. Here, a randomized controlled trial, involving non-human primates, investigated the safety of using FGF-2-CP composite layer-coated screws after either gamma-ray sterilization or aseptic processing. Titanium alloy screws coated with FGF-2-CP composite layers and subjected to either gamma-ray sterilization at 25 kGy (GS group) or aseptic storage (AS group) were implanted into the vertebral bodies of two cynomolgus monkeys exceeding 12 weeks (day 99). Physiological, histological, and radiographic investigations were performed to evaluate the safeties of the screws. There were no serious adverse events, such as surgical site infection, significant loss of body weight, or abnormal blood test results. No radiolucent areas were observed around the screws from the GS or AS group throughout the study. In the intraosseous region, no significant differences were observed in bone and fibrous tissue apposition rates and rate of bone formation between the two groups (p = 0.49, 0.77, and 0.11, respectively). Neither tumor lesions nor accumulation of lymphocytes and neutrophils were observed in either group. Our data suggest that FGF-2-CP composite layer-coated screws subjected to terminal gamma-ray sterilization are as safe as those fabricated in aseptic processing.

Evaluation of tumour necrosis factor alpha-stimulated gene-6 and fibroblast growth factor-2 levels in patients diagnosed with multi-system inflammatory syndrome in children.

Investigations are still ongoing about the pathophysiology of multi-system inflammatory syndrome in children, which can progress with serious morbidity and mortality after COVID-19 infection. In this study, we aimed to investigate whether fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels play a role in the diagnosis of the disease and on cardiac involvement. Twenty-three patients (11 girls, 12 boys) and 26 healthy controls (10 girls, 16 boys) were included in the study. The mean age of the patient and control group was 8.45 ± 2.43 and 10.73 ± 4.27 years, respectively. There was no difference between the fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels of the patient and control groups. When the patients with myocardial involvement in the patient group were compared with the patients without myocardial involvement in terms of fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels, no difference was found between these groups. The correlation of fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels with other laboratory parameters was investigated in the patient group. Fibroblast growth factor-2 was moderately inversely correlated with white blood cell count (r = -0.541, p = 0.008), absolute neutrophil count (r = -0.502, p = 0.015) and C-reactive protein (r = -0.528, p = 0.010). Fibroblast growth factor-2 was strongly inversely correlated with erythrocyte sedimentation rate (r = -0.694, p =<0.001). Our data show that fibroblast growth factor-2 and tumour necrosis factor alpha stimulated gene-6 do not provide sufficient information about diagnosis and cardiac involvement in multi-system inflammatory syndrome in children.

Dual-Warhead Conjugate Based on Fibroblast Growth Factor 2 Dimer Loaded with α-Amanitin and Monomethyl Auristatin E Exhibits Superior Cytotoxicity towards Cancer Cells Overproducing Fibroblast Growth Factor Receptor 1.

Targeting fibroblast growth factor receptor 1 (FGFR1) is a promising therapeutic strategy for various cancers associated with alterations in the FGFR1 gene. In this study, we developed a highly cytotoxic bioconjugate based on fibroblast growth factor 2 (FGF2), which is a natural ligand of this receptor, and two potent cytotoxic drugs-α-amanitin and monomethyl auristatin E-with completely independent mechanistic modes of action. Utilizing recombinant DNA technology, we produced an FGF2 N- to C-end dimer that exhibited superior internalization capacity in FGFR1-positive cells. The drugs were site-specifically attached to the targeting protein using SnoopLigase- and evolved sortase A-mediated ligations. The resulting dimeric dual-warhead conjugate selectively binds to the FGFR1 and utilizes receptor-mediated endocytosis to enter the cells. Moreover, our results demonstrate that the developed conjugate exhibits about 10-fold higher cytotoxic potency against FGFR1-positive cell lines than an equimolar mixture of single-warhead conjugates. The diversified mode of action of the dual-warhead conjugate may help to overcome the potential acquired resistance of FGFR1-overproducing cancer cells to single cytotoxic drugs.

FGF2 and IL-1ß - explorers of unconventional secretory pathways at a glance.

Fibroblast growth factor 2 (FGF2) and interleukin 1ß (IL-1ß) were among the earliest examples of a subclass of proteins with extracellular functions that were found to lack N-terminal secretory signal peptides and were shown to be secreted in an ER- and Golgi-independent manner. Many years later, a number of alternative secretory pathways have been discovered, processes collectively termed unconventional protein secretion (UPS). In the course of these studies, unconventional secretion of FGF2 and IL-1ß were found to be based upon distinct pathways, mechanisms and molecular machineries. Following a concise introduction into various pathways mediating unconventional secretion and transcellular spreading of proteins, this Cell Science at a Glance poster article aims at a focused analysis of recent key discoveries providing unprecedented detail about the molecular mechanisms and machineries driving FGF2 and IL-1ß secretion. These findings are also highly relevant for other unconventionally secreted cargoes that, like FGF2 and IL1ß, exert fundamental biological functions in biomedically relevant processes, such as tumor-induced angiogenesis and inflammation.

Normal aging, motor neurone disease, and Alzheimer's disease are characterized by cortical changes in inflammatory cytokines.

The role of increased brain inflammation in the development of neurodegenerative diseases is unclear. Here, we have compared cytokine changes in normal aging, motor neurone disease (MND), and Alzheimer's disease (AD). After an initial analysis, six candidate cytokines, interleukin (IL)- 4, 5, 6, 10, macrophage inhibitory protein (MIP)-1α, and fibroblast growth factor (FGF)-2, showing greatest changes were assayed in postmortem frozen human superior frontal gyri (n = 12) of AD patients, aging and young adult controls along with the precentral gyrus (n = 12) of MND patients. Healthy aging was associated with decreased anti-inflammatory IL-10 and FGF-2 levels. AD prefrontal cortex was associated with increased levels of IL-4, IL-5, and FGF-2, with the largest increase seen for FGF-2. Notwithstanding differences in the specific frontal lobe gyrus sampled, MND patients' primary motor cortex (precentral gyrus) was associated with increased levels of IL-5, IL-6, IL-10, and FGF-2 compared to the aging prefrontal cortex (superior frontal gyrus). Immunocytochemistry showed that FGF-2 is expressed in neurons, astrocytes, and microglia in normal aging prefrontal cortex, AD prefrontal cortex, and MND motor cortex. We report that healthy aging and age-related neurodegenerative diseases have different cortical inflammatory signatures that are characterized by increased levels of anti-inflammatory cytokines and call into question the view that increased inflammation underlies the development of age-related neurodegenerative diseases.

Fibroblast Growth Factor 2 High Molecular Weight Isoforms in Dentoalveolar Mineralization.

Transgenic mice overexpressing human high molecular weight fibroblast growth factor 2 (HMWFGF2) isoforms in osteoblast and odontoblast lineages (HMWTg) exhibit decreased dentin and alveolar bone mineralization, enlarged pulp chamber, and increased fibroblast growth factor 23 (FGF23). We examined if the alveolar bone and dentin mineralization defects in HMWTg mice resulted from increased FGF23 expression and whether an FGF23 neutralizing antibody could rescue the hypomineralization phenotype. HMWTg and VectorTg control mice were given subcutaneous injections of FGF23 neutralizing antibody twice/week starting at postnatal day 21 for 6 weeks. Since Calcitriol (1,25D) have direct effects in promoting bone mineralization, we also determined if 1,25D protects against the defective dentin and alveolar bone mineralization. Therefore, HMWTg mice were given subcutaneous injections of 1,25D daily or concomitantly with FGF23 neutralizing antibody for 6 weeks. Our results showed that HMWTg mice displayed thickened predentin, alveolar bone hypomineralization, and enlarged pulp chambers. FGF23 neutralizing antibody and 1,25D monotherapy partially rescued the dentin mineralization defects and the enlarged pulp chamber phenotype in HMWTg mice. 1,25D alone was not sufficient to rescue the alveolar bone hypomineralization. Interestingly, HMWTg mice treated with both FGF23 neutralizing antibody and 1.25D further rescued the enlarged pulp chamber size, and dentin and alveolar bone mineralization defects. We conclude that the dentin and alveolar bone mineralization defects in HMWTg mice might result from increased FGF23 expression. Our results show a novel role of HMWFGF2 on dentoalveolar mineralization.

Endothelium-Targeted Deletion of microRNA-15a/16-1 Promotes Poststroke Angiogenesis and Improves Long-Term Neurological Recovery.

RATIONALE: Angiogenesis promotes neurological recovery after stroke and is associated with longer survival of stroke patients. Cerebral angiogenesis is tightly controlled by certain microRNAs (miRs), such as the miR-15a/16-1 cluster, among others. However, the function of the miR-15a/16-1 cluster in endothelium on postischemic cerebral angiogenesis is not known. OBJECTIVE: To investigate the functional significance and molecular mechanism of endothelial miR-15a/16-1 cluster on angiogenesis in the ischemic brain. METHODS AND RESULTS: Endothelial cell-selective miR-15a/16-1 conditional knockout (EC-miR-15a/16-1 cKO) mice and wild-type littermate controls were subjected to 1 hour middle cerebral artery occlusion followed by 28-day reperfusion. Deletion of miR-15a/16-1 cluster in endothelium attenuates post-stroke brain infarction and atrophy and improves the long-term sensorimotor and cognitive recovery against ischemic stroke. Endothelium-targeted deletion of the miR-15a/16-1 cluster also enhances post-stroke angiogenesis by promoting vascular remodeling and stimulating the generation of newly formed functional vessels, and increases the ipsilateral cerebral blood flow. Endothelial cell-selective deletion of the miR-15a/16-1 cluster up-regulated the protein expression of pro-angiogenic factors VEGFA (vascular endothelial growth factor), FGF2 (fibroblast growth factor 2), and their receptors VEGFR2 (vascular endothelial growth factor receptor 2) and FGFR1 (fibroblast growth factor receptor 1) after ischemic stroke. Consistently, lentiviral knockdown of the miR-15a/16-1 cluster in primary mouse or human brain microvascular endothelial cell cultures enhanced in vitro angiogenesis and up-regulated pro-angiogenic proteins expression after oxygen-glucose deprivation, whereas lentiviral overexpression of the miR-15a/16-1 cluster suppressed in vitro angiogenesis and down-regulated pro-angiogenic proteins expression. Mechanistically, miR-15a/16-1 translationally represses pro-angiogenic factors VEGFA, FGF2, and their receptors VEGFR2 and FGFR1, respectively, by directly binding to the complementary sequences within 3'-untranslated regions of those messenger RNAs. CONCLUSIONS: Endothelial miR-15a/16-1 cluster is a negative regulator for postischemic cerebral angiogenesis and long-term neurological recovery. Inhibition of miR-15a/16-1 function in cerebrovascular endothelium may be a legitimate therapeutic approach for stroke recovery.

Comparison of periodontal wound healing/regeneration by recombinant human fibroblast growth factor-2 combined with ß-tricalcium phosphate, carbonate apatite, or deproteinized bovine bone mineral in a canine one-wall intra-bony defect model.

AIM: To evaluate periodontal wound healing/regeneration of one-wall intra-bony defects treated with recombinant human fibroblast growth factor-2 (rhFGF-2) and beta-tricalcium phosphate (ß-TCP), carbonate apatite (CO3 Ap), or deproteinized bovine bone mineral (DBBM) in dogs. MATERIALS AND METHODS: The stability of rhFGF-2 adsorbed onto the bone substitutes was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). One-wall intra-bony defects (5 × 5 × 5 mm) created in five adult male beagle dogs were treated with rhFGF-2 alone (rhFGF-2), rhFGF-2 with ß-TCP (rhFGF-2/ß-TCP), rhFGF-2 with CO3 Ap (rhFGF-2/CO3 Ap), or rhFGF-2 with DBBM (rhFGF-2/DBBM). Histological outcomes (e.g., linear length of new cementum adjacent to the newly formed bone with inserting collagen fibres [NA] as the primary outcome) were evaluated at 10 weeks post surgery. RESULTS: Significantly higher amount of rhFGF-2 was adsorbed onto CO3 Ap compared with ß-TCP. Among the treatment groups, the rhFGF-2/DBBM group showed the highest amount of periodontal tissue regeneration. The rhFGF-2/DBBM group showed significantly greater formation of NA (3.22 ± 0.40 mm) compared with rhFGF-2 (1.17 ± 1.00 mm, p < .01) group. Additionally, new bone area in the rhFGF-2/DBBM group (9.78 ± 2.30 mm2 ) was significantly higher than that in the rhFGF-2 (5.08 ± 1.26 mm2 , p < .01), rhFGF-2/ß-TCP (5.91 ± 1.27 mm2 , p < .05), and rhFGF-2/CO3 Ap (6.51 ± 1.49 mm2 , p < .05) groups. Slight ankylosis was found in the rhFGF-2/ß-TCP (1/9 sites), rhFGF-2/CO3 Ap (3/10 sites), and rhFGF-2/DBBM (1/9 sites) groups. CONCLUSIONS: Within their limitations, the present data indicate that DBBM seems to be a suitable carrier for rhFGF-2 and that rhFGF-2/DBBM treatment promotes favourable periodontal regeneration compared with rhFGF-2, rhFGF-2/ß-TCP, and rhFGF-2/CO3 Ap treatments in one-wall intra-bony defects.