OBJECTIVE

Growth factor based angiogenesis, with or without cell therapy, is a promising therapeutic modality for patients with coronary artery disease. We compared the relative efficacies of surgically delivered vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in a swine model of hypercholesterolemia-induced endothelial dysfunction which captures many of the pathophysiologic abnormalities of human coronary disease.

METHODS

Yucatan mini-swine (20-30 kg), fed a high cholesterol diet (total 20 weeks), underwent circumflex ameroid placement to create chronic myocardial ischemia, followed three weeks later by perivascular administration of VEGF (2 microg; n=6), FGF-2 (100 microg; n=6), or placebo (n=7) in the ischemic territory. Normocholesterolemic animals (n=7) served as controls. Four weeks later, endothelial function, collateral-dependent perfusion, as well as myocardial protein and mRNA levels of angiogenic mediators were assessed.

RESULTS

Endothelial dysfunction was observed in all hypercholesterolemic animals as impaired microvessel relaxation in response to adenosine diphosphate and VEGF. VEGF administration improved baseline-adjusted collateral-dependent perfusion at rest (-0.03+/-0.05 vs -0.12+/-0.04, VEGF vs placebo, p=0.09), but FGF-2 delivery caused a significantly greater improvement in perfusion compared to either group (+0.15+/-0.03, p<0.05 vs HC-placebo and HC-VEGF) at rest. Molecular analysis revealed increased eNOS expression (135%+/-8%, p=0.03 vs placebo) in all growth factor treated animals and increased expression of FGF-2 receptor, FGFR1 (65+/-26%, p=0.04 vs placebo), in FGF-2 treated animals. No significant changes were demonstrated in other angiogenic mediators including Akt, Syndecan-4.

CONCLUSIONS

In the setting of hypercholesterolemic endothelial dysfunction, FGF-2 is more effective than VEGF at enhancing collateral-dependent perfusion and thus, may be a better candidate than VEGF for angiogenic therapy in patients with end-stage CAD.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) induces cell death in cells of the Ewing's sarcoma family of tumors in vivo and in vitro. In this study we demonstrate that this is dependent on the rapid and sustained activation of p38(MAPK), in contrast to the transient activation of p38(MAPK) associated with bFGF-induced cell proliferation. Stem cell <em>factor</em>-induced survival of TC-32 cells was also associated with transient activation of p38(MAPK). Inhibition of p38(MAPK) by SB<em>20</em>2190 and p38(MAPK) small interfering RNA reduces bFGF-induced death in TC-32 cells, consistent with the hypothesis that activation of p38(MAPK) is essential for induction of death by bFGF. This appears to be dependent on sustained activation of p38(MAPK), demonstrated by inhibition of bFGF-induced cell death following addition of SB<em>20</em>2190 to TC-32 cells 5 min after exposure to bFGF (<em>20</em> ng/ml) and activation of p38(MAPK). Prolonged activation of p38(MAPK) is accompanied by a rapid and sustained phosphorylation of Ras and ERK; inhibition of ERK phosphorylation using the MEK-1 inhibitor PD98059 rescued approximately 30% of cells from bFGF-induced death suggesting ERK plays a secondary role in the induction of death. This hypothesis is supported by observations in the A673 cell line; bFGF induced sustained activation of ERK and transient activation of p38(MAPK), which was not associated with cell death. These data demonstrate that sustained activation of p38(MAPK) is essential for activation of the death cascade following exposure of Ewing's sarcoma family of tumors cells to bFGF and provide evidence that activation of p38(MAPK) results in an up-regulation of the death receptor p75(NTR).

OBJECTIVE

To evaluate the effect of conditioned medium from human uterine cervical stem cells (CM-hUCESCs) on corneal epithelial healing in a rat model of dry eye after alkaline corneal epithelial ulcer. We also tested the bactericidal effect of CM-hUCESCs.

METHODS

Dry eye was induced in rats by extraocular lacrimal gland excision, and corneal ulcers were produced using NaOH. Corneal histologic evaluation was made with hematoxylin-eosin (H&E) staining. Real-time PCR was used to evaluate mRNA expression levels of proinflammatory cytokines. We also studied the bactericidal effect of CM-hUCESCs in vitro and on infected corneal contact lenses (CLs) using Escherichia coli and Staphylococcus epidermidis bacteria. In addition, in order to investigate proteins from CM-hUCESCs that could mediate these effects, we carried out a human cytokine antibody array.

RESULTS

After injury, dry eyes treated with CM-hUCESCs significantly improved epithelial regeneration and showed reduced corneal macrophage inflammatory protein-1 alpha (MIP-1α) and TNF-α mRNA expression as compared to untreated eyes and eyes treated with culture medium or sodium hyaluronate ophthalmic drops. In addition, we found in CM-hUCESCs high levels of proteins, such as tissue inhibitors of metalloproteinases 1 and 2, <em>fibroblast</em> <em>growth</em> <em>factor</em> 6 and 7, urokinase receptor, and hepatocyte <em>growth</em> <em>factor</em>, that could mediate these effects. In vitro, CM-hUCESCs showed a clear bactericidal effect on both E. coli and S. epidermidis and CLs infected with S. epidermidis. Analyses of CM-hUCESCs showed elevated levels of proteins that could be involved in the bactericidal effect, such as the chemokine (C-X-C motif) ligands 1, 6, 8, 10, and the chemokine (C-C motif) ligands 5 and <em>20</em>.

CONCLUSIONS

Treatment with CM-hUCESCs improved wound healing of alkali-injured corneas and showed a strong bactericidal effect on CLs. Patients using CLs and suffering from dry eye, allergies induced by commercial solutions, or small corneal injuries could benefit from this treatment.

The involvement of vascular <em>fibroblasts</em> (FBs) and smooth muscle (SM)-like cells in physiological and pathological processes in large vessels (intimal hyperplasia) and microvessels (capillary arterialization), and the realization that these cells are exposed to interstitial flow shear stress (SS), motivate this study of SS on FB migratory activity. Rat adventitial FBs were grown to either 30-50% confluence (subconfluent FBs; SFBs) or full confluence (confluent FBs; CFBs) in culture. Immunofluorescence and Western blotting assays were conducted to evaluate the expression of two phenotype markers: SM alpha-actin and SM myosin heavy chain (MHC). Both assays indicated a significant increase in SM alpha-actin expression in CFBs compared with SFBs, suggesting a phenotype difference between the two cell populations. SFBs and CFBs both expressed minimal SM MHC. Both cell populations were seeded on Matrigel-coated cell culture inserts and exposed to 4 h of either 1 or <em>20</em> dyn/cm(2) SS via a rotating disk apparatus in the presence of the chemoattractant platelet-derived <em>growth</em> <em>factor</em>-BB to quantify the effect of SS on SFB and CFB migration. Four hours of <em>20</em> dyn/cm(2) SS significantly enhanced SFB migration while it suppressed CFB migratory activity. Four hours of 1 dyn/cm(2) SS did not significantly alter either SFB or CFB migration levels. Because of the distinct migratory responses of SFBs and CFBs in response to SS, phenotype modulation appears to be one way to regulate their involvement in both physiological and pathological remodeling processes.

Progressive fibrosis is a pathological hallmark of many chronic diseases responsible for organ failure. Although there is currently no therapy on the market that specifically targets fibrosis, the dynamic fibrogenic process is known to be regulated by multiple soluble mediators that may be therapeutically intervened. The failing hamster heart exhibits marked fibrosis and increased expression of secreted Frizzled-related protein 2 (sFRP2) amenable to reversal by mesenchymal stem cell (MSC) therapy. Given the previous demonstration that sFRP2-null mice subjected to myocardial infarction exhibited reduced fibrosis and improved function, we tested whether antibody-based sFRP2 blockade might counteract the fibrogenic pathway and repair cardiac injury. Cardiomyopathic hamsters were injected intraperitoneally twice a week each with <em>20</em> μg of sFRP2 antibody. Echocardiography, histology, and biochemical analyses were performed after 1 mo. sFRP2 antibody increased left ventricular ejection fraction from 40 ± 1.2 to 49 ± 6.5%, whereas saline and IgG control exhibited a further decline to 37 ± 0.9 and 31 ± 3.2%, respectively. Functional improvement is associated with a ∼ 50% reduction in myocardial fibrosis, ∼ 65% decrease in apoptosis, and ∼ 75% increase in wall thickness. Consistent with attenuated fibrosis, both MSC therapy and sFRP2 antibody administration significantly increased the activity of myocardial matrix metalloproteinase-2. Gene expression analysis of the hamster heart and cultured <em>fibroblasts</em> identified Axin2 as a downstream target, the expression of which was activated by sFRP2 but inhibited by therapeutic intervention. sFRP2 blockade also increased myocardial levels of VEGF and hepatocyte <em>growth</em> <em>factor</em> (HGF) along with increased angiogenesis. These findings highlight the pathogenic effect of dysregulated sFRP2, which may be specifically targeted for antifibrotic therapy.

AMG 416 (velcalcetide), a novel peptide agonist of the calcium-sensing receptor, lowers plasma parathyroid hormone in preclinical uremic animal models and in normal healthy individuals. Here, we studied its efficacy in hemodialysis patients suffering from secondary hyperparathyroidism. Major inclusion criteria were hemodialysis for at least 3 months, serum parathyroid hormone over 300 pg/ml, a corrected serum calcium of 9.0 mg/dl or more, and stable doses of vitamin D analogs for at least 3 weeks prior to screening. Twenty-eight patients were enrolled in one of five cohorts (5, 10, <em>20</em>, 40, 60 mg). Cohorts 1-3 (four patients each) were treated in a two-period crossover design, while cohorts 4 and 5 (eight patients each) were randomized 1:1 to AMG 416 or placebo. Patients were admitted to a clinical research unit following hemodialysis and studied for 3 days prior to discharge for hemodialysis. Single intravenous doses of AMG 416 from 5 to 60 mg were well tolerated, and plasma levels increased in a dose-related manner. AMG 416 treatment was associated with significant, dose-dependent reductions in serum parathyroid hormone and <em>fibroblast</em> <em>growth</em> <em>factor</em> 23. Compared with placebo, all dose groups of 10 mg or more were associated with attenuation in the rise in serum phosphate during the interdialytic period. Dose-dependent reductions in serum calcium were observed and were well tolerated. Thus, AMG 416 represents a novel therapeutic approach for the treatment of secondary hyperparathyroidism in hemodialysis patients.

Systemic and local radiation injuries caused by nuclear power reactor accidents, therapeutic irradiation, or nuclear terrorism should be prevented or properly treated in order to improve wound management and save lives. Currently, regenerative surgical modalities should be attempted with temporal artificial dermis impregnated and sprayed with a local angiogenic <em>factor</em> such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and secondary reconstruction can be a candidate for demarcation and saving the donor morbidity. Human mesenchymal stem cells and adipose-derived stem cells, together with angiogenic and mitogenic <em>factor</em> of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and an artificial dermis, were applied over the excised irradiated skin defect and were tested for differentiation and local stimulation effects in the radiation-exposed wounds. The perforator flap and artificial dermal template with <em>growth</em> <em>factor</em> were successful for reconstruction in patients who were suffering from complex underlying disease. Patients were uneventfully treated with minimal morbidities. In the experiments, the hMSCs are strongly proliferative even after <em>20</em> Gy irradiation in vitro. In vivo, 4 Gy rat whole body irradiation demonstrated that sustained marrow stromal (mesenchymal stem) cells survived in the bone marrow. Immediate artificial dermis application impregnated with cells and the cytokine over the <em>20</em> Gy irradiated skin and soft tissues demonstrated the significantly improved fat angiogenesis, architected dermal reconstitution, and less inflammatory epidermal recovery. Detailed understanding of underlying diseases and rational reconstructive procedures brings about good outcomes for difficult irradiated wound healing. Adipose-derived stem cells are also implicated in the limited local injuries for short cell harvesting and processing time in the same subject.

Human keratinocyte <em>growth</em> <em>factor</em> (KGF) is a recently identified mitogen for epithelial cells produced by normal stromal <em>fibroblasts</em>. KGF has been shown to stimulate keratinocyte migration and promote re-epithelialization of skin suggesting a critical role for KGF in wound healing. To understand how KGF might be regulated during wound healing, we examined the ability of the pro-inflammatory cytokines interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) interleukin-6 (IL-6), tumor necrosis <em>factor</em>-alpha (TNF-alpha) transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) and interferon-gamma (IFN-gamma) to modulate KGF gene expression in cultured human <em>fibroblasts</em>, using northern blot analysis. Exposure to IL-1 alpha (<em>20</em> units/ml) or IL-1 beta (100 units/ml) for 24 h increased KGF mRNA expression by 352% and 504%, respectively, with early induction seen at 2 h and maximal induction seen at 8 h. TNF-alpha (30 ng/ml) increased KGF mRNA expression by 535% at 24 h, with induction first seen at 8 h. The maximal induction of KGF mRNA was observed when IL-1 alpha, IL-1 beta and TNF-alpha were used at 100 units/ml, and 3 ng/ml, respectively, although concentrations 100-500-fold lower (IL-1 alpha, 0.02 units/ml; IL-beta, 0.02 units/ml; and TNF-alpha, 0.03 ng/ml) were nearly as stimulatory, increasing KGF mRNA expression by 175%, 254% and 322%, respectively. IL-6 (<em>20</em>0 units/ml), TGF-beta 1 (5 ng/ml) and IFN-gamma (<em>20</em>0 units/ml) did not change the level of KGF mRNA at 24 h in human <em>fibroblasts</em> under the same conditions. The protein synthesis inhibitor cycloheximide abrogated the effects of IL-1 alpha, IL-1 beta and TNF-alpha on KGF gene induction, indicating that new protein synthesis is required in the process. Dexamethasone (10(-7) M), known to inhibit inflammatory reactions and retard wound healing, also inhibited the induction of KGF mRNA expression by IL-1 alpha, IL-1 beta and TNF-alpha. Individual variation in KGF mRNA expression was see when <em>fibroblasts</em> from different aged donors were analysed, but no consistent age-associated change was observed. These results suggest that IL-1 alpha, IL-1 beta and TNF-alpha up-regulate KGF gene expression in <em>fibroblasts</em> and might be responsible for its induction following skin wounding or other injury.

Ineffectual wound healing in hyperglycaemic patients suffering from diabetes mellitus is characterised by a reduction in capillary reformation (angiogenesis). Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) is secreted by <em>fibroblasts</em>, macrophages and in particular endothelial cells (EC) in response to tissue injury and is important in promotion of neovascularisation. Recently, glycation of FGF-2 has been shown to significantly reduce its activity in vitro. We have examined the kinetics of FGF-2 glycation and compared its ability with that of native FGF-2 to activate mitogenesis, capillary formation and associated signal transduction in bovine aortic EC (BAEC). FGF-2 was exposed to 0.25 M glucose-6-phosphate (G-6-P) for 24-72 h and the degree of glycation determined by matrix assisted laser desorption ionisation mass spectrometry. Native FGF-2 was heterogeneous with Mw in the range 15,153.6-17,903 Da. After 24 h incubation with G-6-P there was evidence of glycation, and the mass increase corresponded to addition of 2.7 mol of G-6-P residues; after 48 h, 4 mol sugar was added and this increased to 8.7 after 72 h. Dimerisation of FGF-2 was observed after 72 h of treatment. Induction of mitogenesis in BAEC was significantly reduced by 25%-40% after treatment for 48-96 h with glycated (24 h) FGF-2 (gFGF-2; 100 pg/ml-5 ng/ml; P < 0.05), whilst capillary tubule formation was significantly reduced by between 60% and 90% (100 pg/ml-1 ng/ml; P < 0.05) after 5 days compared to native FGF-2. Subsequent investigation of the signal transduction molecules associated with mitogenesis showed a reduction in FGF-2 induced tyrosine phosphorylated proteins of approximate Mw <em>20</em>-150 kDa between 10 min and 24 h, in particular, mitogen activated protein kinase (MAPK)/early response kinase (ERK-1, ERK-2), after glycation. To determine the reason for reduced angiogenic activity of gFGF-2, we compared its binding characteristics to that of native FGF-2. Total binding of gFGF-2 to the cell surface was significantly reduced in BAEC analysed by FACS compared to native FGF-2 (P < 0.05). Further investigation using 125I-labelled differentially washed samples, demonstrated a significant reduction in gFGF-2 binding to the high affinity tyrosine kinase receptor (46%) compared to native FGF-2. In summary, glycation of FGF-2 in vitro occurs rapidly within 24 h in the presence of elevated levels of G-6-P. Glycation caused a significant reduction in the ability of FGF-2 to bind to the tyrosine kinase receptor and activate signal transduction pathways responsible for both mitogenesis and capillary formation in BAEC. These results could help to explain the mechanism behind impaired wound healing in patients with diabetes mellitus.

To comprehensively screen for genetic events underlying colorectal cancer, we performed suppression subtraction hybridization analysis on an advanced colon cancer. Because Dickkopf-4, a member of the Dickkopf family acting as a Wnt-signaling modulator, was identified as one of the upregulated genes in this specimen, we investigated expression profiles of all the Dickkopf family members in 55 colorectal tumors (21 cancers and 34 adenomas). We also investigated mechanisms regulating the expression of Dickkopf-4 in these cancers in vitro and in vivo. Compared with normal adjacent mucosae, Dickkopf-4 (median 27.4, P < 0.01) and -2 (median 51.4, P < 0.01) were strongly expressed in colorectal cancers. The level of Dickkopf-4 was positively correlated with <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>20</em> (r(s) = 0.61, P = 0.00017), a representative beta-catenin transcriptional target gene, and with the degree of nuclear accumulation of beta-catenin in colorectal tumors. Dickkopf-4 was induced by activated beta-catenin in vitro. Reciprocally, recombinant Dickkopf-4 significantly inhibited T-cell <em>factor</em>/lymphocyte enhancer <em>factor</em> reporter activity stimulated by recombinant Wnt3a in human embryonic kidney 293 cells. We conclude that Dickkopf-4 and -2 are significantly upregulated in most colorectal tumors, and that Dickkopf-4 upregulation reflects activation of the Wnt/canonical pathway.

BACKGROUND

Diabetic nephropathy imposes a substantial cardiovascular and renal burden contributing to both morbidity and excess mortality. Progression of chronic kidney disease (CKD) in diabetes mellitus is variable, and few biomarkers are available to predict progression accurately. Identification of novel predictive biomarkers may inform clinical care and assist in the design of clinical trials. We hypothesized that urinary and plasma protein biomarkers predict CKD progression independently of the known clinical markers such as albuminuria and estimated glomerular filtration rate (eGFR) in diabetic nephropathy.

METHODS

We studied 67 US veterans with CKD due to type 2 diabetes mellitus and <em>20</em> age-matched controls (no CKD, hypertension or cardiovascular disease). After clinical evaluation and the collection of blood and urine specimens for 24 biomarkers, we followed subjects prospectively for the next 2-6 years. CKD progression was defined in three ways: (i) clinically by examining eGFR versus time plots for each individual (slope progression), (ii) progression to end-stage renal disease (ESRD) and (iii) a composite outcome of ESRD or death.

RESULTS

Among 17 urinary and 7 plasma biomarkers evaluated, the relationship of the biomarkers with outcome was as follows: (i) for progression identified by eGFR plots, urinary C-terminal fibroblast growth factor (FGF)-23 emerged to have the strongest primary association (adjusted odds ratio [aOR] 2.08, P = 0.008); (ii) for ESRD, plasma vascular endothelial growth factor (VEGF) had an association (aOR: 1.44, P = 0.027) and (iii) for the composite outcome of death and ESRD, plasma C-terminal FGF-23 also had a robust direct association (aOR: 3.07, P = 0.008).

CONCLUSIONS

The relationship of biomarkers with future progression of CKD is complex and depends in part on how CKD progression is defined. Biomarkers in the FGF-23 and VEGF-A pathways predicted patient progression independently of albuminuria levels in this patient cohort. Additional studies in other cohorts will help further validate this pilot study.

Adipose tissue is an abundant source of autologous adult stem cells that may bring new therapeutic perspectives on the treatment of diabetes and its complications. It is unclear whether adipose tissue-derived stromal cells (ASCs) of diabetic patients, constantly influenced by hyperglycaemia, have the same properties as non-diabetic controls. As an alternative source of ASCs, adipose tissue from distal limbs of diabetic patients with critical ischemia was isolated. ASCs were characterized in terms of cell surface markers, multilineage differentiation and the expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGFA), chemokine-related genes and compared with non-diabetic controls. Flow cytometry analysis confirmed mesenchymal phenotypes in both diabetic and non-diabetic ASCs. Nevertheless, 40% of diabetic and <em>20</em>% of non-diabetic ASC samples displayed high expressions of <em>fibroblast</em> marker, which inversely correlated with the expression of CD105. In diabetic patients, significantly decreased expression of VEGFA and chemokine receptor CXCR4 was found in <em>fibroblast</em>-positive ASCs, compared with their <em>fibroblast</em>-negative counterparts. Reduced osteogenic differentiation and the downregulation of chemokine CXCL12 were found in <em>fibroblast</em>-negative diabetic ASCs. Both diabetic and non-diabetic ASCs were differentiated into adipocytes and chondrocytes and did not reveal islet-like cell differentiation. According to this study, adipose tissue from distal limbs of diabetic patients is not satis<em>factor</em>y as an autologous ASC source. Hyperglycaemic milieu as well as other metabolic disorders linked to diabetes may have an influence on endogenous stem cell properties. The present study investigated the feasibility of autologous stem cell therapy in diabetic patients. ASCs isolated from the ischemic limb of diabetic patients were found to be less potent when compared phenotypically and functionally to control non-diabetic counterparts with no signs of limb ischemia. High expression of <em>fibroblast</em> markers associated with reduced expression of VEGFA as well as reduced osteogenic differentiation may have an impact on the effectiveness of autologous cell therapies in diabetic patients.

Compound K (CK; <em>20</em>-O-beta-D-glucopyranosyl-<em>20</em>(S)-protopanaxadiol), an active ginseng saponin metabolite, exerts anticancer activity via apoptosis induction in various cancers. In the present study, we investigated the anti-angiogenic activity of CK and its molecular mechanisms in human umbilical vein endothelial cells (HUVECs). Angiogenesis was induced in HUVECS by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a potent angiogenic <em>growth</em> <em>factor</em>. CK significantly inhibited the proliferation and also attenuated the expression of a proliferating protein cyclin D1 in bFGF treated HUVECs. Also, CK significantly inhibited the migration and tube formation of bFGF treated HUVECs at non-cytotoxic concentrations, reduced secreted level of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and increased the secreted level of pigment epithelium-derived <em>factor</em> (PEDF) in HUVECs. In addition, CK effectively disrupted bFGF-induced neo-vascularization in the Matrigel plugs excised from mice in vivo. Notably, we have found that CK downregulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and AKT in bFGF treated HUVECs. Taken together, our findings for the first time indicate that CK exerts anti-angiogenic activity via inhibition of p38 MAPK and AKT in HUVECs with the potential of a cancer chemopreventive agent.

The sensory and supporting cells (SCs) of the organ of Corti are derived from a limited number of progenitors. The mechanisms that regulate the number of sensory progenitors are not known. Here, we show that <em>Fibroblast</em> <em>Growth</em> <em>Factors</em> (FGF) 9 and <em>20</em>, which are expressed in the non-sensory (Fgf9) and sensory (Fgf<em>20</em>) epithelium during otic development, regulate the number of cochlear progenitors. We further demonstrate that Fgf receptor (Fgfr) 1 signaling within the developing sensory epithelium is required for the differentiation of outer hair cells and SCs, while mesenchymal FGFRs regulate the size of the sensory progenitor population and the overall cochlear length. In addition, ectopic FGFR activation in mesenchyme was sufficient to increase sensory progenitor proliferation and cochlear length. These data define a feedback mechanism, originating from epithelial FGF ligands and mediated through periotic mesenchyme that controls the number of sensory progenitors and the length of the cochlea.

Recent advances in understanding the biology of fracture healing and the availability of specific macromolecules has resulted in the development of novel treatments for injuries to bone. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (4 mg/ml), a potent mitogen, and hyaluronan (<em>20</em> mg/ml), an extracellular matrix component, were combined into a viscous gel formulation intended for direct, percutaneous injection into fresh fractures. In an experimental primate fracture model, a bilateral 1-mm-gap osteotomy was surgically created in the fibulae of baboons. A single direct administration of this hyaluronan/<em>fibroblast</em> <em>growth</em> <em>factor</em>-2 formulation to the defect site significantly promoted local fracture healing as evidenced by increased callus formation and mechanical strength. Radiographic analysis showed that the callus area was statistically significantly larger at the treated sites than at the untreated sites. Specimens treated with 0.1, 0.25, and 0.75 ml hyaluronan/<em>fibroblast</em> <em>growth</em> <em>factor</em>-2 demonstrated a 48, 50, and 34% greater average load at failure and an 82, 104, and 66% greater energy to failure than the untreated controls, respectively. By histologic analysis, the callus size, periosteal reaction, vascularity, and cellularity were consistently more pronounced in the treated osteotomies than in the untreated controls. These results suggest that hyaluronan/<em>fibroblast</em> <em>growth</em> <em>factor</em>-2 may provide a significant advance in the treatment of fractures.

Dentin matrix protein-1 (DMP1) or phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) inactivation results in elevation of the phosphaturic hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-23, leading to hypophosphatemia, aberrant vitamin D metabolism, and rickets/osteomalacia. Compound mutant Phex-deficient Hyp and Dmp1(ko) mice exhibit nonadditive phenotypes, suggesting that DMP1 and PHEX may have interdependent effects to regulate FGF23 and bone mineralization. To determine the relative importance of DMP1 and PHEX in regulating FGF23 and mineralization, we tested whether the transgenic expression of full-length [Dmp1(Tg(full-length))] or C-terminal Dmp1 [Dmp1(Tg(57kDa))] could rescue the phenotype of Hyp mice. We found that Dmp1(ko) and Hyp mice have similar phenotypes characterized by decreased cortical bone mineral density (-35% vs. wild type, P < 0.05) and increased serum FGF23 levels (~12-fold vs. wild type, P < 0.05). This was significantly corrected by the overexpression of either the full-length or the C-terminal transgene in Dmp1(ko) mice. However, neither of the transgenes rescued the Hyp mice phenotype. Hyp/Dmp1(Tg(full-length)) and Hyp mice were similar, but Hyp/Dmp1(Tg(57 kDa)) mice exhibited worsening of osteomalacia (-<em>20</em>% cortical bone mineral density) in association with increased serum FGF23 levels (+2-fold) compared with Hyp mice. Bone FGF23 mRNA expression was decreased and a 2-fold increase in the ratio of the full-length/degraded circulating FGF23 was observed, indicating that degradation of FGF23 was impaired in Hyp/Dmp1(Tg(57 kDa)) mice. The paradoxical effects of the C-terminal Dmp1 transgene were observed in Hyp/Dmp1(Tg(57 kDa)) but not in Dmp1(Tg(57 kDa)) mice expressing a functional PHEX. These findings indicate a functional interaction between PHEX and DMP1 to regulate bone mineralization and circulating FGF23 levels and for the first time demonstrate effects of the C-terminal DMP1 to regulate FGF23 degradation.

Vitamin D deficiency, diagnosed when the serum 25-hydroxyvitamin D (25-OHD(3)) concentration is less than <em>20</em> ng/mL, has joined vitamin A deficiency as two of the most common nutrition-responsive medical conditions worldwide. There have been more scientific articles published about vitamin D in the 21st century than about any other vitamin, reflecting the massive expansion of the field of vitamin D research. Adequate vitamin D status has been linked to decreased risks of developing specific cancers, including cancers of the esophagus, stomach, colon, rectum, gallbladder, pancreas, lung, breast, uterus, ovary, prostate, urinary bladder, kidney, skin, thyroid, and hematopoietic system (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma); bacterial infections; rheumatoid arthritis; Crohn's disease; periodontal disease; multiple sclerosis; asthma; type 2 diabetes; cardiovascular disease; stroke; peripheral artery disease; hypertension; chronic kidney disease; muscle weakness; cognitive impairment; Alzheimer's disease; clinical depression; and premature death. On the other hand, inadequate vitamin D status during human pregnancy may be associated with increased risk for the development of type 1 diabetes in the offspring. However, this point of view may be excessively optimistic. There also is evidence that despite the current heavy reliance on serum 25-OHD(3) concentration for the diagnosis of an individual's vitamin D status, local tissue vitamin D intoxication may be present in individuals with much lower serum 25-OHD(3) concentrations than are currently appreciated. Only rarely are the symptoms of local tissue vitamin D intoxication associated with vitamin D status or intake. An individual's serum 25-OHD(3) concentration may appear to be "low" for reasons totally independent of sunlight exposure or vitamin D intake. Serum 25-OHD(3) concentration is only poorly responsive to increases in vitamin D intake, and the prolonged routine consumption of thousands of international units of vitamin D may interfere with the regulation of phosphate homeostasis by <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF23) and the Klotho gene product, with consequences that are detrimental to human health. In light of these counterbalancing observations, curbing excessive enthusiasm for universally increasing vitamin D intake recommendations may be in order.

BACKGROUND

Persistent upregulation of signaling by cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) through its receptor fibroblast growth factor-inducible molecule-14 (Fn14) promotes chronic inflammation and tissue destruction.

OBJECTIVE

The aim of this study was to explore the safety and tolerability of the TWEAK-blocking monoclonal antibody BIIB023 and determine its pharmacokinetics and effects on TWEAK pathway pharmacodynamic markers in rheumatoid arthritis (RA).

METHODS

Phase I, first-in-human, 2-part, multicenter, double-blind, dose-escalation study. Patients were randomized to a single dose of BIIB023 (0.03-20 mg/kg) (n = 38) or placebo (n = 15) as an add-on to methotrexate. Three open-label cohorts of RA patients taking background disease-modifying antirheumatic drugs and stable tumor necrosis factor (TNF) inhibitor therapy (n = 12) received a single-dose of BIIB023 of 2, 10, or 20 mg/kg and were assessed over 70 days.

RESULTS

The incidence of treatment-emergent adverse events for the BIIB023 monotherapy cohorts and open-label cohorts of BIIB023 as add-on therapy to TNF inhibitors compared with placebo were 47% and 50% versus 33%, respectively. Serum exposure to BIIB023 increased in a dose-dependent manner from 0.03 to 20 mg/kg, but not in direct proportion to dose level. After administration, the time course of BIIB023 serum concentration was multiphasic and showed expedited elimination when levels decreased to < 10 µg/mL. Serum-soluble TWEAK levels were suppressed at all dose levels by 6 hours post-dose and recovered to baseline between days 7 and 28. A trend toward downward modulation of serum biomarkers of inflammatory response was suggested in monocyte chemoattractant protein 1, inducible protein 10, macrophage inflammatory protein 1β, and tissue inhibitor of metalloproteinase 1 in the BIIB023 group versus placebo.

CONCLUSIONS

Single-dose BIIB023 showed a favorable safety and tolerability profile in RA. Suppression of serum-soluble TWEAK for ≤ 28 days was observed and downward trends in serum biomarkers suggested.

OBJECTIVE

Disordered mineral metabolism is a common complication of chronic kidney disease (CKD) and a novel risk factor for CKD progression, cardiovascular disease, and mortality. Although diabetes is the leading cause of CKD and is associated with worse clinical outcomes than other etiologies, few studies have evaluated mineral metabolism in CKD according to diabetes status.

METHODS

Using the Chronic Renal Insufficiency Cohort Study, we tested the hypothesis that diabetes is independently associated with lower serum calcium and higher serum phosphate, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23).

RESULTS

Compared with participants without diabetes (n = 1,936), those with diabetes (n = 1,820) were more likely to have lower estimated glomerular filtration rate (eGFR), lower serum albumin, and higher urinary protein excretion (all P < 0.001). Unadjusted serum phosphate, PTH, and FGF23 levels were higher and calcium was lower among those with compared with those without diabetes (all P < 0.001). After multivariate adjustment, diabetes remained a significant predictor of serum phosphate, PTH, and FGF23 but not calcium. The eGFR cut point at which 50% of participants met criteria for secondary hyperparathyroidism or elevated FGF23 was higher in participants with diabetes compared with those without (PTH: eGFR 30-39 vs. 20-29, P < 0.001; FGF23: eGFR 50-59 vs. 40-49, P < 0.001).

CONCLUSIONS

Disordered mineral metabolism begins earlier in the course of CKD and is more severe among CKD patients with compared with those without diabetes. Future studies should explore mechanisms for these differences and whether they contribute to excess risks of adverse clinical outcomes among diabetic patients with CKD.

<AbstractText>Obesity and type 2 diabetes mellitus (T2DM) are risk <em>factors</em> for nonalcoholic fatty liver disease, including nonalcoholic steatohepatitis. This study assessed pegbelfermin (BMS-986036), recombinant PEGylated human <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21), in patients with obesity and T2DM predisposed to fatty liver.</AbstractText><p><div><b>METHODS</b></div>In this randomized, double-blind, placebo-controlled study, patients with T2DM and BMI of 30 to 50 kg/m² received subcutaneous pegbelfermin (1, 5, or <em>20</em> mg daily or <em>20</em> mg weekly; n = 96) or placebo (n = 24) for 12 weeks. Primary end points were safety, tolerability, and change in HbA1c. Additional end points included insulin sensitivity, lipids, adiponectin, and disease progression biomarkers.</p><AbstractText>There were no significant effects of pegbelfermin versus placebo on HbA1c. Pegbelfermin <em>20</em> mg/d significantly improved high-density lipoprotein cholesterol (P = 0.015) and triglycerides (P = 0.037). All pegbelfermin regimens significantly increased adiponectin levels; <em>20</em>-mg daily and weekly regimens decreased serum PRO-C3. Most adverse events were mild; the most frequent adverse events were injection-site bruising and diarrhea.</AbstractText><AbstractText>Twelve-week pegbelfermin treatment did not impact HbA1c concentrations, but QW and higher daily doses were associated with improved metabolic parameters and fibrosis biomarkers in patients with obesity and T2DM predisposed to fatty liver. These results support evaluation of pegbelfermin in patients with obesity-related metabolic diseases (e.g., nonalcoholic steatohepatitis).</AbstractText>

OBJECTIVE

Peptide growth factors are known accelerators of corneal wound healing, probably mediated through increased proliferation of the cells; however, information about their effect on keratocyte motility is lacking. The influence of peptide growth factors on keratocyte migratory activity was investigated, using the following growth factors: platelet derived growth factor (PDGF-BB), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and transforming growth factor-beta-1 (TGF-beta 1).

METHODS

Keratocytes were seeded on gels of type 1 collagen, growth factor added, and the cells left to migrate for 72 hours. Subsequently, the number of keratocytes at the different levels in the collagen gel was evaluated by optically sectioning the gel at 20 microns, intervals, with an inverted phase contrast microscope.

RESULTS

PDGF, EGF and bFGF at 10 ng/ml, all increased the number of keratocytes at the different levels of the gel as compared to a non-stimulated control (p < 0.05 or p < 0.01, students t-test). TGF-beta proved to be a strong inhibitor of keratocyte migration, decreasing the number of keratocytes observed at every level in the gel (p < 0.05 and p < 0.01, students t-test), whereas no effect of IGF-I and aFGF was found. During the 72 hours of migration, no contraction of the collagen gels was observed. Autoradiography of histological sections of the gels showed that during the 72-hour period only TGF-beta and 10% fetal bovine serum induced an increase in keratocyte proliferation.

CONCLUSIONS

PDGF, EGF and bFGF increase keratocyte migration, independent of proliferation in a collagen gel invasion assay and might promote corneal wound healing, not only by increasing cell proliferation, but also through increased motility.

Patients with biliary tract cancers (cholangiocarcinoma (CCA) and gallbladder cancer (GBC)) have a poor prognoses and incidence is increasing. Most patients are diagnosed with advanced disease when options of treatment are limited to palliative approaches, mainly focused on chemotherapy. In recent years, novel treatment targets of relevance to biliary tract cancers, mainly present in patients with intrahepatic cholangiocarcinoma (iCCA), have been identified and are rapidly changing the field. These include <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) fusions and isocitrate dehydrogenase (IDH)-1 and -2 mutations which are each present in around 10-<em>20</em>% of patients with iCCA patients. In addition, inhibition of other pathways is currently being explored, such as human epidermal <em>growth</em> <em>factor</em> receptor (HER) family, the Wnt pathway, neurotropic tyrosine kinase receptor (NTRK) fusions and BRAF mutations. The IDH1 inhibitor ivosidenib has already been tested in a phase III clinical trials in pre-treated CCA and showed benefit in terms of progression-free survival (the primary end-point). Multiple FGFR inhibitors are currently in development in phase II and III trials. There has been a consistently high response rate reported for these compounds (ranging between <em>20</em>.7% and 35.5%), especially for patients harbouring FGFR2 fusions. This review provides an overview of the status of targeted therapies in biliary tract cancers. Detailed discussion regarding current clinical development of IDH and FGFR inhibitors is provided, with an overview of current caveats and future steps.

BACKGROUND

Aqueous flare as determined by laser flare photometry in the anterior chamber is a strong preoperative predictor for proliferative vitreoretinopathy (PVR) in patients with primary retinal detachment (RD). We analysed various cytokines in aqueous humour samples in relation to aqueous flare and postoperative PVR incidence in patients with RD.

METHODS

Preoperatively, the aqueous flare of patients with RD was measured quantitatively with a laser flare metre and aqueous humour samples were collected and analysed for interferon γ, tumour necrosis factor α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, vascular endothelial growth factor (VEGF)-A, platelet derived growth factor (PDGF)-aa, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, fibroblast growth factor (FGF)-aa and FGF-bb by multiplex fluorescent bead-based immunoassays. Three months after RD surgery patients were examined for PVR development.

RESULTS

Of 67 consecutive patients, 10 developed at least PVR grade C. Patients with flare values >15 pc/ms (n=20) and the 10 patients with postoperative PVR all had significantly elevated levels of IL-6, IL-8, MCP-1 and TGF-β1 in aqueous humour (p≤0.05). Levels of VEGF-A, PDGF-aa and TGF-β2 were not significantly changed. Other cytokines were below the detection threshold. Eight of the 10 patients (80%) with PVR had elevated flare values of >15 pc/ms and 8 of the 20 patients (40%) with flare >15 pc/ms developed PVR. The OR for PVR with flare values >15 pc/ms was 30.7 (p=0.0001).

CONCLUSIONS

Laser flare photometry allows simple risk estimation for later PVR development. Elevated laser flare values correspond to an altered profibrotic intraocular cytokine milieu. These factors therefore constitute promising targets for a prophylactic intervention.

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and <em>fibroblasts</em> using CMP-coated dishes. The CMP coating at 10-<em>20</em> micrograms/ml enhanced the adhesion and spreading of rabbit <em>growth</em> plate, resting and articular chondrocytes, and <em>fibroblasts</em> and human epiphyseal chondrocytes and MRC5 <em>fibroblasts</em>. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion <em>factor</em> for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion <em>factor</em>, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.