We have studied insulin binding to cultured skin <em>fibroblasts</em> from a patient with the Type A syndrome of insulin resistance and acanthosis nigricans. Insulin binding was decreased aobut 50% at low insulin concentrations. This was due to a decrease in receptor affinity and in increase in the rate of dissociation of insulin from the receptor. In addition, there was a loss of negative cooperativity, as measured by the ability of unlabeled insulin to accelerate dissociation. This defect in the receptor was stable for up to <em>16</em> passages of the cells. By contrast, binding of epidermal <em>growth</em> <em>factor</em> did not differ from control. These data suggest that the Type A syndrome of insulin resistance is due to a primary, and possibly genetic, defect in the insulin receptor.

A new method for culturing retinal Muller cells from adult bovine tissue is described. The identification of these glial cells was based on immunocytochemical analysis of specific Muller cell markers. Cultured cells from fourth to ninth passage showed positive labelling for S 100 protein, carbonic anydrase (CAA), glutamine synthetase (GS), alpha cristallin (alpha C) and polyclonal glial fibrillary acidic protein (GFAP) antibody, but were negative for both monoclonal GFAP antibody and also for Muller cells in the retina. Investigation of the effect of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and epithelial <em>growth</em> <em>factor</em> (EGF) on the proliferation of the Muller cells revealed that bFGF was the most potent mitogen (EC50 = 14 pM). Binding data revealed the presence of two classes of binding sites for aFGF and bFGF: (1) a high affinity binding site (Kd of 14 pM and 27 pM for aFGF and bFGF respectively); (2) a low affinity binding site (Kd of 3.2 nM and 0.6 nM for aFGF and bFGF respectively with great variability in the number of binding sites). In addition, the cross-linking experiments revealed the presence of high molecular weight FGF receptors (110-140 kDa). After aFGF or bFGF binding to Muller cells, aFGF and bFGF-cell surface receptors were rapidly downregulated with a half-life for disappearance of 35-50 min. Internalization and degradation of 125I-bFGF bound to the Muller cell receptors did not occur at 4 degrees C. At 37 degrees C, however, there was a rapid decrease in receptor-bound 125I-bFGF due to the downregulation of bFGF receptors. Concomitantly 125I-bFGF appeared inside the Muller cells. After 2 h, 125I-bFGF began to be degraded and after 6 h three fragments of <em>16</em> kDa, 8 kDa and 5.5 kDa were discernible. Degradation of bFGF appeared to occur in the lysosomal compartment since it was inhibited by chloroquine, an inhibitor of lysosomal proteases; aFGF internalization and degradation followed the same kinetics as bFGF with the appearance of 7 kDa and 5 kDa fragments. These results suggest that Muller cells may be the target for aFGF and bFGF contained in other cells of the retina. The fact that aFGF could be released from rod outer segment by a phosphorylation-dependent mechanism, and that apical prolongation of the Muller cells is connected with the photoreceptor cells suggest that these <em>factors</em> may be the mediators involved in the communication between glial cells and neurons.

Stromal-derived <em>factor</em> 1 (SDF-1) and CXCR4 comprise a unique chemokine/chemokine receptor pair, exhibiting important functions in morphogenesis and <em>growth</em> regulation as well as attractant properties on T lymphocytes. No data are available on SDF-1 and CXCR4 in normal or pathological thyroid tissues. SDF-1, CXCR4, and CD18 messenger ribonucleic acid (mRNA) as a marker of leukocytic infiltration were quantified in tissues affected by thyroid adenoma (n = 11) and Graves' disease (GD; n = <em>16</em>) using competitive RT-PCR. SDF-1 mRNA levels differed significantly between autonomous adenomas and the corresponding normal tissue, but not in GD between patients with low or high leukocyte infiltration, thyroid peroxidase, and TSH receptor autoantibodies, respectively. We found a strong correlation between CXCR4 and CD18 mRNA, which indicates CXCR4 expression by leukocytes. To define the cellular source of SDF-1 and CXCR4 in thyroid tissue, we examined various thyroid-derived cells. <em>Fibroblasts</em> are the most potent producers of SDF-1, although thyrocytes also secrete SDF-1 in vitro. Leukocytes showed very weak SDF-1 mRNA levels and no secretion of the chemokine. Immunohistology confirmed and extended these results; SDF-1 expression was found in <em>fibroblasts</em>, but not or very weakly in CD45(+) leukocytes and thyrocytes. Only leukocytes were CXCR4(+). As examined by flow cytometry, the number of CD3(+) T cells expressing CXCR4 is significantly higher in the thyroid than in peripheral blood. SDF-1 seems to be involved in thyroid tissue homeostasis in thyroid adenoma, but not in the maintenance of lymphocytic infiltration in GD.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) play important roles during fetal and embryonic development. FGF-2 (basic FGF, bFGF) is widely expressed in the embryo and has been linked to tissue <em>growth</em> and remodeling. However, it is tightly bound to heparin sulfate proteoglycans of the extracellular matrix which quenches its biological activity. We showed previously that a secreted FGF-binding protein (FGF-BP) can mobilize and activate FGF-2 from the extracellular matrix. While considerable data exist on the expression and pivotal role of FGF-BP in tumor <em>growth</em>, less is known about FGF-BP during embryonic development. In this immunohistochemical study in mice, we show FGF-BP protein expression in a broad spectrum of tissues at various stages between day 8 and day <em>16</em> of embryonal development, and compare FGF-BP and FGF-2 immunolocalization. FGF-BP is detected in the digestive system, thymus, skin, hair follicles, dental germ, respiratory tract, various glandular tissues, kidney, liver, and certain areas of the CNS, with immunoreactivity being mainly confined to cells of primitive epithelia. The putative significance of these findings with regard to mobilization of FGF-2 or other molecules is discussed. From the locally confined, time-dependent, and apparently tightly regulated FGF-BP expression we propose that FGF-BP may play an important role in embryonic development.

OBJECTIVE

Excessive scarring following trauma or surgery of cornea, conjunctiva or retina can greatly impair visual outcome. At present, no agents are clinically available that selectively reduce activity of genes that regulate fibrosis. Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues, including cornea and conjunctiva. In this study, hammerhead ribozymes targeting CTGF mRNA were synthesized, kinetic parameters were measured, and the effect on TGF-beta-mediated cell proliferation was measured in cultured human fibroblasts.

METHODS

The mRNA sequence of human CTGF was scanned for potential hammerhead ribozyme cleavage sites, and predicted secondary folding structures around the sites were calculated. Synthetic 12mer ribozymes and 33mer oligonucleotide mRNA targets corresponding to two sites were synthesized, and kinetic constants calculated from Hanes-Wolff plots of in vitro cleavage reactions. The ribozyme with higher percentage cleavage and kinetic rate was cloned into an expression plasmid (pTR-UF21) and stably transfected into cultured human fibroblasts. An inactive ribozyme plasmid served as a negative control. The effects of the ribozyme on expression of TGF-beta-induced CTGF mRNA and protein levels were measured using ELISA and real-time TaqMan quantitative RT-PCR. Finally, the effect of the CTGF ribozyme on TGF-beta-mediated proliferation of fibroblasts was measured using a non-radioactive cell proliferation microtiter assay.

RESULTS

Of the eight potential hammerhead ribozyme cleavage sites in human CTGF mRNA, two sites (CHR 745, and CHR 859) were identified with optimal secondary folding. CHR 859 cleaved 94% of the target mRNA, compared to 46% cleavage for CHR 745 after 16 hr of reaction. CHR 859 had a K(m) of 1.56 microM and a K(cat) of 2.97 min(-1), while CHR 745 had a K(m) of 7.80 microM and a K(cat) of 5.7 min(-1). The turnover numbers (K(cat)/K(m)) of CHR 859 and CHR 745 were 1.9 x 10(6) M min(-1) and 7.4 x 10(5) M min(-1), respectively, indicating CHR 859 is 2.6 times more efficient than CHR 745 in destroying CTGF mRNA. Stable transfection of CHR 859 into human fibroblasts reduced CTGF mRNA levels 55% and protein levels 72% compared to the inactive ribozyme control. Furthermore, TGF-beta-induced cell proliferation was reduced 90% in fibroblasts stably transfected with CHR 859 compared to control cell groups.

CONCLUSIONS

The CHR 859 hammerhead ribozyme cleaved human CTGF mRNA with high kinetic efficiency in vitro, effectively reduced levels of CTGF mRNA and protein in cultured human fibroblasts, and blocked TGF-beta-induced cell proliferation without nonspecific toxicity. These data support the concept that CTGF mediates TGF-beta-induced cell proliferation, and imply that regulating CTGF expression with ribozymes may be effective in reducing ocular scarring.

Effects of a moderate-intensity static magnetic field (SMF) on the early-stage development of endothelial capillary tubule formation were examined during the initial cell <em>growth</em> periods using co-cultured human umbilical vein endothelial cells and human diploid <em>fibroblasts</em>. The co-cultured cells within a well (<em>16</em> mm in diameter) were exposed to SMF intensity up to 120 mT (Bmax) with the maximum spatial gradient of 21 mT/mm using a disc-shaped permanent magnet (<em>16</em> mm in diameter and 2.5 mm in height) for up to 10 days. Control exposure was performed without magnet. Some vascular endothelial cells were treated with vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-A (10 ng/ml) to promote the tubule formation every 2-3 days. Four experimental protocols were performed: (1) non-exposure (control); (2) SMF exposure alone; (3) non-exposure with VEGF-A; (4) SMF exposure with VEGF-A. Photomicrographs of tubule cells immunostained with an anti-platelet-endothelial cell adhesion molecule-1 (PECAM-1 [CD31[) antibody as a pan-endothelial marker, were analyzed after culture at 37 degrees C for 4, 7, and 10 days. The mean values of the area density and the length of tubules (related mainly to arteriogenesis) as well as the number of bifurcations (related mainly to angiogenesis) were determined as parameters of tubule formation and were compared between the groups. After a 10 day incubation, in the peripheral part of the culture wells, SMF alone significantly promoted the tubule formation in terms of the area density and the length of tubules, compared with control group. In the central part of the wells, however, SMF did not cause any significant changes in the parameters of tubule formation. After a 7 day incubation, VEGF-A significantly promoted all the parameters of tubule formation in any part of the wells, compared with control group. With regard to the synergistic effects of SMF and VEGF-A on tubule formation, after a 10 day incubation, SMF significantly promoted the VEGF-A-increased area density and length of tubules in the peripheral part of the wells, compared with the VEGF-A treatment alone. However, SMF did not induce any significant changes in the VEGF-A-increased number of bifurcations in any part of the wells. The tubule cells observed in the wells had elongated, spindle-like shapes, and the direction of cell elongation was random, irrespective of the presence and direction of SMF. These findings suggest that the application of SMF to intact or VEGF-A-stimulated vascular endothelial cells leads mainly to promote or enhance arteriogenesis in the peripheral part of the wells, where the spatial gradient increases relative to the central part. The effects of SMF on the VEGF-A-enhanced tubule formation appear to be synergistic or additive in arteriogenesis but not in angiogenesis.

OBJECTIVE

Cinacalcet and vitamin D are often combined to treat secondary hyperparathyroidism (SHPT) in patients on dialysis. Independent effects on fibroblast growth factor-23 (FGF-23) concentrations in patients on hemodialysis administered cinacalcet or vitamin D analogs as monotherapies during treatment of SHPT are evaluated.

METHODS

A multicenter, randomized, open-label study to compare the efficacy of cinacalcet versus traditional vitamin D therapy for management of secondary hyperparathyroidism among subjects undergoing hemodialysis (PARADIGM) was a prospective, phase 4, multicenter, randomized, open-label study conducted globally. Participants (n=312) were randomized 1:1 to cinacalcet (n=155) or vitamin D analog (n=157) for 52 weeks. Levels of FGF-23 were measured at baseline and weeks 20 and 52. The absolute and percentage changes from baseline in plasma FGF-23, parathyroid hormone (PTH), calcium (Ca), phosphorus (P), and calcium-phosphorus product (Ca×P) were assessed. Correlations and logistic regression were used to explore relationships between changes in FGF-23 and changes in PTH, Ca, P, and Ca×P from baseline to week 52 by treatment arm.

RESULTS

Median (quartiles 1, 3) decrease in FGF-23 concentrations was observed in the cinacalcet arm (-40%; -63%, 16%) compared with median increase in the vitamin D analog arm (47%; 0%, 132%) at week 52 (P<0.001). Changes in FGF-23 in both arms were unrelated to changes in PTH (cinacalcet: r=0.17, P=0.11; vitamin D analog: r=-0.04, P=0.70). Changes in FGF-23 in the vitamin D analog but not the cinacalcet arm were correlated with changes in Ca (cinacalcet: r=0.11, P=0.30; vitamin D analog: r=0.32, P<0.01) and P (cinacalcet: r=0.19, P=0.07; vitamin D analog: r=0.49, P<0.001). Changes in FGF-23 were correlated with changes in Ca×P in both arms (cinacalcet: r=0.26, P=0.01; vitamin D analog: r=0.57, P<0.001). Independent of treatment arm, participants with reductions in P or Ca×P were significantly more likely to show reductions in FGF-23.

CONCLUSIONS

During treatment of SHPT, cinacalcet use was associated with a decrease in FGF-23 concentrations, whereas vitamin D analogs were associated with an increase. The divergent effects of these treatments on FGF-23 seem to be independent of modification of PTH. It is possible that effects of cinacalcet and vitamin D analogs on FGF-23 may be mediated indirectly by other effects on bone and mineral metabolism.

Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing (RNA-Seq) of poly(A) and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and <em>16</em> h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response <em>factors</em>, and components of the unfolded protein response (UPR) was observed. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm of the UPR was activated, as the expression of both activating transcription <em>factor</em> 4 (ATF4) and CHOP (DDIT3), critical regulators of the pathway, was altered after infection. Expression of the transcription <em>factor</em> early <em>growth</em> response 1 (EGR1) was induced in a PERK-dependent manner. EGR1(-/-) mouse embryonic <em>fibroblasts</em> (MEFs) demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection.

OBJECTIVE

Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 (EGR1) contributed to VEEV-induced cell death.

Systemic inflammation and mitochondrial dysfunction are involved in neurodegeneration in Parkinson's disease (PD). Extracellular vesicle (EV) trafficking may link inflammation and mitochondrial dysfunction. In the present study, circulating small EVs (sEVs) from <em>16</em> older adults with PD and 12 non-PD controls were purified and characterized. A panel of serum inflammatory biomolecules was measured by multiplex immunoassay. Protein levels of three tetraspanins (CD9, CD63, and CD81) and selected mitochondrial markers (adenosine triphosphate 5A (ATP5A), mitochondrial cytochrome C oxidase subunit I (MTCOI), nicotinamide adenine dinucleotide reduced form (NADH):ubiquinone oxidoreductase subunit B8 (NDUFB8), NADH:ubiquinone oxidoreductase subunit S3 (NDUFS3), succinate dehydrogenase complex iron sulfur subunit B (SDHB), and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2)) were quantified in purified sEVs by immunoblotting. Relative to controls, PD participants showed a greater amount of circulating sEVs. Levels of CD9 and CD63 were lower in the sEV fraction of PD participants, whereas those of CD81 were similar between groups. Lower levels of ATP5A, NDUFS3, and SDHB were detected in sEVs from PD participants. No signal was retrieved for UQCRC2, MTCOI, or NDUFB8 in either participant group. To identify a molecular signature in circulating sEVs in relationship to systemic inflammation, a low level-fused (multi-platform) partial least squares discriminant analysis was applied. The model correctly classified 94.2% ± 6.1% PD participants and 66.7% ± 5.4% controls, and identified seven biomolecules as relevant (CD9, NDUFS3, C-reactive protein, <em>fibroblast</em> <em>growth</em> <em>factor</em> 21, interleukin 9, macrophage inflammatory protein 1β, and tumor necrosis <em>factor</em> alpha). In conclusion, a mitochondrial signature was identified in circulating sEVs from older adults with PD, in association with a specific inflammatory profile. In-depth characterization of sEV trafficking may allow identifying new biomarkers for PD and possible targets for personalized interventions.

Colony-stimulating <em>factor</em> 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, <em>growth</em>, or differentiation of the inner cell mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and <em>growth</em> of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal <em>fibroblasts</em> if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE out<em>growth</em>s from blastocysts. The exception was a decrease in out<em>growth</em> size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9-<em>16</em> cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE out<em>growth</em>s. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB.

The effect on intraosseous bone formation of a single local injection of recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> into trabecular bones was examined in ovariectomised osteoporotic rats. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (400 micrograms), or the vehicle alone, was injected into the ilium at <em>16</em> weeks after ovariectomy or a simulated operation. Bone mineral density in the ovariectomised rats increased to a level similar to the latter at 2 weeks and reached a maximum at 8 weeks. After 8 weeks, BMD decreased slowly and the value at 24 weeks was still higher than that in the ovariectomised rats. <em>Fibroblast</em> <em>growth</em> <em>factor</em> stimulated osteoid formation in the first 2 weeks, bone volume reaching a peak at 8 weeks. From 8 to 12 weeks, bone resorption increased, resulting in decreases in bone volume to the levels of the group with simulated operations at 24 weeks. Structural analysis at 8 and 24 weeks showed that ovariectomy decreased the continuity of trabeculae and the injection of <em>fibroblast</em> <em>growth</em> <em>factor</em> restored it to levels higher than, or equal to, those who had the simulated operation. The present study demonstrated that intraosseous <em>fibroblast</em> <em>growth</em> <em>factor</em> given to ovariectomised rats restored bone volume and quality to the levels of the rats who had a simulated operation only.

Inflammatory bowel disease consists of Crohn's disease (CD) and ulcerative colitis (UC). A major clinical problem in some patients is to differentiate clearly between these entities, which is important when planning appropriate medical and surgical treatment. Connective tissue <em>growth</em> <em>factor</em> (CTGF), a novel peptide involved in fibrotic disorders, was analyzed in the present study in CD and UC patients to evaluate its possible role in these two disorders. Twenty-five normal human intestinal tissue samples were obtained through an organ donor program. CD tissues were obtained from 28 individuals undergoing partial intestinal resection (17 small bowel; 11 large bowel) due to complications of the disease. UC tissue samples were obtained from <em>16</em> patients undergoing colectomy due to complications of the disease. Expression of CTGF was studied by Northern blot analysis. In situ hybridization was used to localize mRNA moieties in the tissue samples. Northern blot analysis revealed an average 5-fold increase in CTGF mRNA expression in 89% (25/28) of CD tissue samples by comparison with normal controls (p < 0.0001). In contrast, in UC samples CTGF mRNA levels were comparable to those of normal controls. However, UC tissue samples exhibited enhanced TGF-beta1 mRNA levels (4-fold; p < 0.05). In situ hybridization in CD samples showed CTGF mRNA localized especially in <em>fibroblasts</em> within the submucosal layer, around lymph follicles and in some areas of intense damage in the proximity of the luminal surface, whereas inflammatory cells were devoid of any CTGF mRNA signal. The present data indicate that CTGF plays a different role in IBD and might be useful, especially in those cases with unusual disease presentation, to better differentiate UC and CD. In addition, our data indicate a crucial role for CTGF in CD, where fibrosis and stenosis are frequent complications that require surgery.

OBJECTIVE

To analyze the effects of application of 1% and 3% insulin-like growth factor I (IGF-1) cream on the process of wound healing in induced skin lesions in diabetic and non-diabetic rats and evaluate its effect on expression of myofibroblasts.

METHODS

Ninety-six Wistar adult male rats were divided into six groups, with 16 rats in each group, as follows: group 1: non-diabetic, untreated; group 2: non-diabetic, treated with 1% IGF-1 cream; group 3: non-diabetic, treated with 3% IGF-1 cream; group 4: diabetic, untreated; group 5: diabetic, treated with 1% IGF-1 cream; and group 6: diabetic, treated with 3% IGF-1 cream. In groups 4, 5, and 6, diabetes was induced by intravenous injection of alloxan. After diabetes had been induced, animals were mantained for 3 months. The experimental procedure consisted of the creation of a circular incision of 0.9 mm in diameter using a metal punch. Following this, wounds were treated daily according to the assigned treatment regimen. Groups 2 and 5 were treated with 1% IGF-1 cream, groups 3 and 6 with 3% IGF-1 cream, and groups 1 and 4 and the untreated groups with 0.9% saline solution. From each group, samples from 4 rats were taken at three, seven, 14, and 21 days after the injury. Samples were fixed in 10% formalin to prepare slides for histological analysis. Slides stained with hematoxylin-eosin (H&E) and Masson were observed vascular proliferation, mononuclear cells, polymorphonuclear cells, fibroblast proliferation, re-epithelialization, and collagen fibers. This study analyzed the expression of α-smooth muscle actin using specific antibodies to correlate the temporal expression of α-smooth muscle-specific actin (α-SM actin), a molecular marker for myofibroblast transformation.

RESULTS

Macroscopic observation of wounds showed a more rapid re-epithelialization of wounds treated with IGF. Regarding acute inflammatory reactions, the results of the analysis of vascular proliferation and polymorphonuclear and mononuclear cells showed no statistically significant differences in any of the periods studied (according to the results of a Mann-Whitney test). The initial immunohistochemical analysis of tissue samples conducted to compare the expression of α-smooth muscle actin between groups showed a relevant response in the expression of myofibroblasts. Data were analyzed using ANOVA and were found to be statistically significant.

CONCLUSIONS

The topical application of 1% and 3% IGF-1 creams increases the expression of myofibroblasts in the process of wound healing in rats.

Syndecan-1 (CD138), a cell-surface heparan sulfate proteoglycan, is involved in cell-cell, cell-matrix interaction and <em>growth</em> <em>factor</em> binding. Loss of expression of syndecan-1 in tumor cells leads to decreased intercellular cohesion, increased potential for tumor invasiveness, and metastatic spread. Furthermore, induction of syndecan-1 expression in the tumor stroma has been postulated to promote tumor angiogenesis via its binding to <em>growth</em> <em>factors</em> such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Although syndecan-1 expression within tumor cells has been investigated in head and neck squamous cell carcinoma, stromal expression has not been studied in detail. We analyzed 38 cases of head and neck squamous cell carcinoma by immunohistochemical staining for syndecan-1 expression within the stroma. The expression of syndecan-1 within tumor cells of various histologic grades of differentiation, squamous cell carcinoma in situ cells, and benign squamous epithelium was also determined. Variable levels of diminished syndecan-1 expression were noted within the dysplastic cells of 9 of <em>16</em> (60%) squamous cell carcinoma in situ lesions and in all 38 (100%) invasive squamous cell carcinoma. In general, higher levels of syndecan-1 expression were observed in the well-differentiated tumors, in contrast to significant reduction of expression seen in poorly differentiated tumors. Syndecan-1 expression was observed within the stroma (in <em>fibroblasts</em>) surrounding infiltrating carcinoma cells in 28 of 38 (74%) cases. The intensity of syndecan-1 staining within the stroma showed generally an inverse correlation with the degree of tumor cell differentiation. Syndecan-1 expression was not detected in the stroma beneath normal squamous epithelium or adjacent to areas of squamous cell carcinoma in situ. We conclude that induced expression of syndecan-1 in the stroma surrounding tumor cells of invasive head and neck squamous cell carcinoma is a frequent event. The increased stromal syndecan-1 expression, coupled with its loss from the surface of carcinoma cells, may contribute to tumor cell invasion and the development of metastases.

OBJECTIVE

Upregulation of matrix metalloproteinase (MMP) and vascular endothelial growth factor (VEGF) has been suggested to have an important role in the pathogenesis of nasal polyps (NPs). The aim of this study was to investigate the effect of rhinovirus (RV) infection on the expression of MMPs, tissue inhibitor of metalloproteinase (TIMP)-1, and VEGF in NP fibroblasts.

METHODS

NP fibroblasts (5 x 10(5) cells/mL) obtained from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) were infected with RV serotype 16 (RV-16) for 4 hours. The RV-16 infection was confirmed by seminested reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. After 48 hours, MMP-2, MMP-9, TIMP-1, and VEGF protein levels were measured from culture supernatants by enzyme-linked immunosorbent assay. The changes in the expression of MMP-2, MMP-9, TIMP-1, and VEGF mRNA were assayed by RT-PCR.

RESULTS

RV-16 infection significantly enhanced the gene and protein expressions of MMP-2, MMP-9, and VEGF in NP fibroblasts, whereas TIMP-1 expression was not significantly affected by RV-16. MMP-2, MMP-9, and VEGF protein expression increased by 2.39-, 2.99-, and 3.02-fold, respectively, in RV-infected NP fibroblasts compared to noninfected controls. RV-16 infection also significantly upregulated the expression of MMP-2, MMP-9, and VEGF mRNA by 1.27-, 1.70-, and 1.53-fold, respectively, compared to control levels.

CONCLUSIONS

These in vitro findings suggest that RV infection may contribute to the pathogenesis of NP formation in patients with CRSwNP.

Nanosized drug carriers functionalized with moieties specifically targeting tumor cells are promising tools in cancer therapy, due to their ability to circulate in the bloodstream for longer periods and their selectivity for tumor cells, enabling the sparing of healthy tissues. Because of its biocompatibility, high bioresorbability, and responsiveness to pH changes, synthetic biomimetic nanocrystalline apatites are used as nanocarriers to produce multifunctional nanoparticles, by coupling them with the chemotherapeutic drug doxorubicin (DOXO) and the DO-24 monoclonal antibody (mAb) directed against the Met/Hepatocyte <em>Growth</em> <em>Factor</em> receptor (Met/HGFR), which is over-expressed on different types of carcinomas and thus represents a useful tumor target. The chemical-physical features of the nanoparticles are fully investigated and their interaction with cells expressing (GTL-<em>16</em> gastric carcinoma line) or not expressing (NIH-3T3 <em>fibroblasts</em>) the Met/HGFR is analyzed. Functionalized nanoparticles specifically bind to and are internalized in cells expressing the receptor (GTL-<em>16</em>) but not in the ones that do not express it (NIH-3T3). Moreover they discharge DOXO in the targeted GTL-<em>16</em> cells that reach the nucleus and display cytotoxicity as assessed in an MTT assay. Two different types of ternary nanoparticles are prepared, differing for the sequence of the functionalization steps (adsorption of DOXO first and then mAb or vice versa), and it is found that the ones in which mAb is adsorbed first are more efficient under all the examined aspects (binding, internalization, cytotoxicity), possibly because of a better mAb orientation on the nanoparticle surface. These multifunctional nanoparticles could thus be useful instruments for targeted local or systemic drug delivery, allowing a reduction in the therapeutic dose of the drug and thus adverse side effects. Moreover, this work opens new perspectives in the use of nanocrystalline apatites as a new platform for theranostic applications in nanomedicine.

In the present study, we compare expression, storage and secretion of the atrial natriuretic <em>factor</em> (ANF) in atrial and ventricular adult rat cardiomyocytes (aARC and vARC) in long-term culture. The influence of insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) and of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) on ANF production and secretion, as well as on the expression of a structural component, alpha-smooth muscle actin (alpha-sm actin), was studied in the two cell types. Antibodies against alpha-ANF were used for immunocytochemical localization of ANF. aARC contained more ANF-granules than vARC, and they were distributed throughout the cell bodies. Quantitative determination of ANF storage and secretion was done by radioimmunoassay (RIA; 125I), and it was demonstrated that aARC stored and secreted ANF 18- and <em>16</em>-times more, respectively, when compared to vARC. Immuno-electron microscopy confirmed that ANF storing secretory granules were present in both types of cardiomyocytes. Expression of ANF and alpha-sm actin in aARC and vARC responded differently to treatment with either IGF-I or bFGF. In aARC, neither IGF-I nor bFGF had an influence on expression of ANF. In vARC, expression of ANF was downregulated by IGF-I and upregulated by bFGF with regard to both immunoreactivity and message. In contrast to vARC, expression of alpha-sm actin was not affected by IGF-I in aARC, whereas bFGF produced a strong upregulation similar to that found in vARC. Mitogen-activated protein kinases (MAPK) 42 and 44, though, were equally activated by bFGF and IGF-I in both aARC and vARC.

BACKGROUND

Extracellular vesicles (EVs), including circulating microvesicles (MVs) or mi- croparticles (MPs) and exosomes, derived from cells or platelets are present in the peripheral blood and are important elements involved in the activation of the coagulation system, transport of macromolecules and intercellular communication. In patients with vascular complications (including diabetes), the number of EVs is significantly increased during the acute phase of the disease. However, less is known about EVs release in the chronic state of diabetes.

OBJECTIVE

To analyse the profile of inflammatory cytokines and angiogenic factors in EVs in diabetic patients with ocular and vascular complications.

METHODS

The study included patients with diabetes and varying degrees of ocular complications including retinopathy (n = 48) and the control group (n = 13). EV-enriched and EV-depleted fractions were obtained from platelet-poor plasma by means of the centrifugation method (16 000 g, for 90 min). In screening, the profile of cytokines with pro-angiogenic effects was preliminary assessed using the protein microarray technology for controlled diabetic patients - CD, uncontrolled diabetic patients - UD and for the control group. In all patients, concentrations of cytokines: RANTES (Regulated on Activation, Normal T-cell Expressed and secreted) and Ang-2 (angiopoietin-2) were assayed using the ELISA method. Common blood and biochemical tests were performed.

RESULTS

In patients with diabetes, analysis of supernatant revealed significantly increased concentrations of basic fibroblast growth factor (bFGF) and soluble receptor for vascular endothelial growth factor 2 (V-EGFR2) when compared to the control group: 49 (10.5-122) vs. 24 (2-72.5) SD (p = 0.03) and 260 (195.5-351) vs. 360 (256-461.5) SD (p = 0.01). In UD patients, concentrations of RANTES, angiostatin, tumor necrosis factor-α (TNF), and tissue inhibitors of metalloproteinase 1 and 2 (TIMP1 and TIMP2) were relatively higher in the EV-enriched fraction when compared to the EV-depleted fraction. Post hoc analysis revealed significant differences between UC patients and the control group in RANTES (16.73 (14.41-18.93) vs. 14.62 (12.37-15.28) mg/ml; p = 0.0235) and Ang-2 (2.76 (2.23-4.64) ng/ml vs. 1.74 (1.54-1.93); p = 0.0316) concentrations. These analyses did not reveal any significant differences in RANTES and Ang-2 concentrations between CD patients and the control group.

CONCLUSIONS

The profiles of cytokines and angiogenic factors in EVs are significantly increased in patients with diabetes. Also, the formation of specific cytokines related to EVs is strongly influenced by disease duration and successful treatment. EVs seem to be the conveyors of upregulated cytokines and angiogenic agents in diabetic patients.

In the ovary, the development of new capillaries from pre-existing ones (angiogenesis) is a complex event regulated by numerous local <em>factors</em>. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial <em>growth</em> <em>factor</em> (VEGF), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), insulin-like <em>growth</em> <em>factor</em> (IGF), angiopoietin (ANPT) and hypoxia-inducible <em>factor</em> (HIF) family members. Antral follicles in our study were classified according to the oestradiol-17-beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5-5, 5-20, 20-180 and >180 ng/ml FF). The corresponding sizes of follicles were 5-7, 8-10, 10-13, 12-14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12 13-<em>16</em> and >18 of the oestrous cycle and months 1-2, 3-4, 6-7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT-qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF-A, FGF-2, IGF-1 and IGF-2, ANPT-2/ANPT-1 and HIF-1-alpha was found during final follicle maturation and in CL during the early luteal phase (days 1-4) followed by a lower plateau afterwards. The results suggest the importance of these <em>factors</em> for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.

<AbstractText>Despite improved outcomes associated with immunotherapies for non-small cell lung cancer (NSCLC), many patients do not respond to treatment. Therefore, there is still an unmet need for molecularly targeted therapies in this patient population. Fusions of the RET oncogene have been identified as driver alterations in patients with NSCLC. Lenvatinib is a multityrosine kinase inhibitor of vascular endothelial <em>growth</em> <em>factor</em> receptors 1-3, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors 1-4, RET, and other targets. This study evaluated the safety and efficacy of lenvatinib in patients with RET fusion-positive lung adenocarcinoma.</AbstractText><AbstractText>In this phase 2, multicenter, open-label study (NCT01877083), patients with RET-positive lung adenocarcinoma received oral lenvatinib 24 mg/day. The primary end point was objective response rate (ORR) by investigator review per Response Evaluation Criteria In Solid Tumors v1.1 criteria. The secondary end points included safety and tolerability, progression-free survival (PFS), and overall survival (OS).</AbstractText><AbstractText>Of 536 patients who screened for study inclusion and exclusion, 25 patients with RET translocations (KIF5B-RET [n = 13] and CCDC6-RET [n = 12]) were identified and received lenvatinib. The overall ORR was <em>16</em>% (95% CI: 4.5%-36.1%). At data cutoff (February 3, 20<em>16</em>), the median PFS was 7.3 months (95% CI: 3.6-10.2) and the median OS was not reached. Duration of response was not estimable at the time of data cutoff. All patients experienced a treatment-emergent adverse event (TEAE); 23 (92%) patients experienced a TEAE of ≥ grade 3, and 6 (24%) patients discontinued lenvatinib due to a TEAE. The most common TEAEs were hypertension (68%), nausea (60%), decreased appetite (52%), diarrhea (52%), and proteinuria (48%).</AbstractText><AbstractText>Lenvatinib demonstrated activity in patients with RET fusion-positive lung adenocarcinomas; although the response rate was relatively low, the median PFS supports the activity of lenvatinib in these patients.</AbstractText>

Although regorafenib has demonstrated survival benefits in patients with metastatic colorectal and gastrointestinal stromal tumors, no proven biomarker has been identified for predicting sensitivity to regorafenib. Here, we investigated preclinical activity of regorafenib in gastric and colorectal cancer cells to identify genetic alterations associated with sensitivity to regorafenib. Mutation profiles and copy number assays of regorafenib target molecules indicated that amplification of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) was the only genetic alteration associated with in vitro sensitivity to regorafenib. Regorafenib effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules in a dose-dependent manner and selectively in FGFR2-amplified cells. Regorafenib induced G1 arrest (SNU-<em>16</em>, KATO-III) and apoptosis (NCI-H7<em>16</em>); however, no significant changes were seen in cell lines without FGFR2 amplification. In SNU-<em>16</em> mice xenografts, regorafenib significantly inhibited tumor <em>growth</em>, proliferation, and FGFR signaling compared to treatment with control vehicle. Regorafenib effectively abrogates activated FGFR2 signaling in FGFR2-amplified gastric and colorectal cancer and, therefore, might be considered for integration into treatment in patients with FGFR2-amplified gastric and colorectal cancers.

It has been shown that the E5 protein of the human papillomavirus type <em>16</em> modulates epidermal <em>growth</em> <em>factor</em> receptor downregulation in monolayer cultures of human keratinocytes and mouse <em>fibroblasts</em>. We have now analysed the effect of this protein on the expression, the distribution and the activation of EGF receptors in raft cultures derived from an E5-transfected human keratinocyte cell line. The epithelia generated in these cultures were stratified and exhibited suprabasal expression of cytokeratins 1 and 10, which are known markers of early epidermal differentiation. In situ hybridization with an antisense riboprobe to the human papilloma virus type <em>16</em> E5 protein revealed a homogeneous gene expression within the entire epithelium of E5-transfected but not empty vector-transfected control cultures. Treatment of serum-starved rafts with EGF for 48 hours led to a strong decrease of suprabasal EGF receptors in control cultures, but not in rafts of E5-expressing cells. Under these conditions, no activated receptors were observed in control cultures, but activated receptors were still present in E5-raft cultures. Our results indicate that human papilloma virus type <em>16</em> E5-mediated modulation of EGF receptor expression occurs in a time- and structure-dependent manner in epithelial equivalents of human keratinocytes.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) exerts anabolic actions on bone formation. Here we investigated the potential effects of recombinant human FGF-2 (rhFGF-2) on the repair process of osteonecrosis of the femoral head (ONFH) and the development of secondary osteoarthritis (OA) in adult rabbits. ONFH was induced by intramuscular injection with methylprednisolone, and vascular occlusion of the capital femoral epiphysis by electrocoagulation, in adult Japanese white rabbits. Animals were randomized into two groups: treatment and control. The treatment group was given a single local injection into the femoral head of 100 μg rhFGF-2 in 100 μl gelatin hydrogel microspheres 8 weeks after the ONFH procedure, and the control group was given phosphate-buffered saline in 100 μl gelatin hydrogel microspheres. Morphological, histopathological, and radiologic analyses, including micro-computed tomography scans and magnetic resonance imaging, showed collapse of the femoral head and progression of articular cartilage degeneration in the control group at <em>16</em> weeks after the single local injection of rhFGF-2. In contrast, rhFGF-2 treatment resulted in new bone formation in the femoral head and prevented the femoral head from collapsing. In addition, the changes in OA, assessed by the modified Mankin score, was significantly lower in the treatment group. Our results indicate that a single local injection of rhFGF-2 microspheres promoted the repair of the osteonecrotic femoral head and inhibited femoral head collapse and OA progression. rhFGF-2 may be a promising strategy for the treatment of ONFH.

Angiogenesis, the process of new blood vessel formation, is important in wound healing, inflammation, tumorigenesis and metastases. During this process, it is a critical step of the loosening of cellular interactions between endothelial cells, which are dependent on the architecture of adherens junction constructed by homophilic interactions of cell surface cadherins. Several studies suggested that the dynamic changes of cadherins are necessary during angiogenesis. However, the mechanism of cadherins regulation on endothelial cells requires further delineation. Here, we showed that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a pivotal pro-angiogenic <em>factor</em>, can downregulate typical cadherins (E-, N-, P- and VE-cadherin) expression on the surface of human umbilical vein endothelial cells (HUVECs) via FGF receptor 1 (FGFR1) signaling. The bFGF-mediated surface cadherin downregulation was significantly reversed only when the HUVECs were treated with JNK inhibitor (SP600125), but not ERK (PD98059) or p38 inhibitor (SB203580). Infecting HUVECs with a dominant negative H-Ras mutant (Ras(S17N)) interferes bFGF-mediated cadherin downregulation, and the result suggests that bFGF attenuates surface cadherin expression on HUVECs via FGFR1 and intracellular Ras-JNK signaling. However, after <em>growth</em> <em>factors</em> withdrawal, FGFR1 blockade or JNK inhibition for <em>16</em> h, cadherins were re-expressed on cell surface of HUVECs. But the mRNA or total protein of cadherins had no significant change, suggesting that the effect of bFGF on cadherin expression may work through a post-translational control. Our data first suggest that JNK participates in bFGF-mediated surface cadherin downregulation. Loss of surface cadherins may affect the cell-cell interaction between endothelial cells and facilitate angiogenesis.