This study was carried out to investigate the expression of various <em>growth</em> <em>factors</em> (GFs) involved in mucosal healing and thereby to clarify whether there are potential differences in the expression of GFs between normal colonic mucosa and the uninvolved mucosa of ulcerative colitis (UC). GF mRNA was investigated by reverse transcription polymerase chain reaction in colorectal biopsies from 20 normal controls and <em>15</em> UC patients. The positive rates (%) for mRNA expression for normal/UC were: epidermal <em>growth</em> <em>factor</em> (EGF) 65/53, transforming <em>growth</em> <em>factor</em> (TGF)-alpha 100/87, TGF-beta 1 60/33, insulin like <em>growth</em> <em>factor</em>-I 45/33, platelet-derived <em>growth</em> <em>factor</em>-A 55/67, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> 0/0, hepatocyte <em>growth</em> <em>factor</em> (HGF) 50/53, EGF receptor 20/27, erb-B2 75/73, and HGF receptor (c-MET) 55/67. Semiquantitation of mRNA showed significantly lower expression of TGF-beta 1 (P < 0.05) in UC. Differences in expression and mRNA levels were not statistically significant for any other GFs. Our results indicate that mucosa in chronic persistent UC has a low basal expression of TGF-beta 1 mRNA, and, since TGF-beta 1 is a multifunctional GF that plays important roles in regulating repair and regeneration following tissue injury, this low expression may be partially responsible for the intractability of the disease.

OBJECTIVE

The development of fibrotic diseases is associated with alterations in the transforming growth factor beta (TGF-beta) pathway. We have investigated the expression and activity of Smad transcription factors of the TGF-beta pathway in primary tunical fibroblasts derived from patients with Peyronie's disease and from controls.

METHODS

Primary fibroblasts were established from biopsies obtained from plaques of 16 patients with Peyronie's disease or the tunica albuginea of 8 control patients. The expression and activity of Smad transcription factors in control and TGF-beta-stimulated primary fibroblasts were investigated at the RNA and protein level by reverse transcription-polymerase chain reaction, Western blotting, and immunofluorescence.

RESULTS

RNA expression levels of Smad3 and Smad4 were significantly increased in fibroblasts from patients with Peyronie's disease. When stimulated with TGF-beta1, fibroblasts showed rapid nuclear translocation of Smad2/3, as soon as 15 min after stimulation. This effect was more pronounced and exhibited an earlier onset in fibroblasts from patients with Peyronie's disease, compared with controls. In addition, an increased nuclear retention time of Smad4 was observed in fibroblasts from patients with Peyronie's disease.

CONCLUSIONS

The expression and activity of Smad transcription factors of the TGF-beta pathway is increased in fibroblasts of patients with Peyronie's disease. Alterations in the TGF-beta pathway seem to be a pathogenetic factor in the development of Peyronie's disease.

Gammadelta T-lymphocytes are believed to play a role in maintaining the normal configuration of epithelial tissue. As little is known about the <em>factors</em> mediating this function, we addressed the question of whether gammadelta T-lymphocytes produce <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-9 as well as two other <em>growth</em> <em>factors</em> associated with epithelial tissue reconstitution. Blood gammadelta T cells isolated from healthy donors were grown in the presence of isopentenyl pyrophosphate (IPP) or transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1)/interleukin-<em>15</em> (IL-<em>15</em>) for 24 h and were assessed for the expression and synthesis of FGF-9, keratinocyte <em>growth</em> <em>factor</em> (KGF), and epidermal <em>growth</em> <em>factor</em> (EGF). Resting human gammadelta T cells constitutively expressed KGF and FGF-9 mRNA but no EGF mRNA. In the presence of IPP, FGF-9 mRNA expression significantly increased in a dose-dependent manner, expression of KGF remained unaltered, and EGF mRNA could not be detected. In contrast to IPP, stimulation of the cells with TGF-beta1/IL-<em>15</em> did not alter FGF-9 expression. Moreover, stimulation with anti-CD3 does not induce FGF-9 expression but triggers a high signal of interferon-gamma mRNA. Western blot analysis of gammadelta T cell lysates, prepared 4 days following stimulation with IPP, showed an increase of FGF-9 protein as compared with control cells. In conclusion, the results demonstrate for the first time that human blood and bronchoalveolar lavage gammadelta T-lymphocytes are capable of expressing FGF-9. The data also provide novel evidence that immunoregulatory cells can synthesize FGF-9.

Periodontitis is a frequently diagnosed oral disease characterized by bone resorption and soft tissue loss around teeth. Unfortunately, currently available therapies only slow or arrest progress of the disease. Ideally, treatment of periodontal defects should be focused on complete regeneration of the lost tissues [(bone and periodontal ligament (PDL)]. As a result, this study used intrabony defects to evaluate the regenerative potential of an injectable macroporous calcium phosphate cement (CaP) in combination with bone morphogenetic protein-2 (BMP-2) or <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2). After creating 30 periodontal defects in <em>15</em> Wistar rats, three treatment strategies were conducted: application of CaP only, CaP + BMP-2 and CaP + FGF-2. Animals were euthanized after 12 weeks and processed for histology and histomorphometry. Using CaP alone resulted in limited effects on PDL and bone healing. CaP + BMP-2 showed a good response for bone healing; a significant 2.4 fold increase in bone healing score was observed compared to CaP. However, for PDL healing, CaP + BMP-2 treatment showed no difference compared to the CaP group. The best results were observed with the combined treatment of CaP + FGF-2, which showed a significant 3.3 fold increase in PDL healing score compared to CaP + BMP-2 and a significant 2.6 fold increase compared to CaP. For bone healing, CaP + FGF-2 showed a significant 1.9 fold increase compared to CaP but no significant difference was noted compared to the CaP + BMP-2 group. The combination of a topical application of FGF-2 and an injectable CaP seems to be a promising treatment modality for periodontal regeneration.

The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein-polysaccharide binding is still debated. The <em>15</em> paracrine <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans.

Pancreatic <em>fibroblasts</em> are continuously gaining ground as an important component of tumor microenvironment that dynamically interact with cancer cells to promote tumor progression. In addition, these tumor-infiltrated <em>fibroblasts</em> can acquire an activated phenotype and produce excessive amounts of extracellular matrix creating a highly dense stroma, a situation known as desmoplasia. Desmoplasia, along with the uncontrolled proliferation of cancer cells, leads to the development of compressive forces within the tumor, generating the so-called solid stress. Solid stress is previously shown to affect cancer cell proliferation and migration, however there is no pertinent study taking into account the effects of solid stress on <em>fibroblasts</em> and whether these effects contribute to tumor progression. In this work, we applied a defined compressive stress on pancreatic <em>fibroblasts</em>, similar in magnitude to that experienced by cells in native pancreatic tumors. Our results suggest that solid stress stimulates <em>fibroblasts</em> activation and strongly upregulates <em>Growth</em> Differentiation <em>Factor</em>-<em>15</em> (GDF<em>15</em>) expression. Moreover, co-culture of compression-induced activated <em>fibroblasts</em> with pancreatic cancer cells significantly promotes cancer cell migration, which is inhibited by shRNA-mediated silencing of GDF<em>15</em> in <em>fibroblasts</em>. Conclusively, our findings highlight the involvement of biophysical <em>factors</em>, such as solid stress, in tumor progression and malignancy revealing a novel role for GDF<em>15</em>.

OBJECTIVE

Cardiac rupture is a catastrophic complication that occurs after acute myocardial infarction (MI) and, at present, there are no effective pharmacological strategies for preventing this condition. Here we investigated the effect of the angiotensin II receptor blocker olmesartan (Olm) on post-infarct cardiac rupture and its underlying mechanisms of action.

METHODS

C57Bl/6 mice with MI were treated with Olm, aldosterone (Aldo) or vehicle. Cultured neonatal cardiomyocytes and fibroblasts were exposed to normoxia or anoxia and treated with angiotensin II (Ang II), RNH6270 (active ingredient of Olm) or Aldo.

RESULTS

The mortality rate and incidence of cardiac rupture in MI mice during the first week in the Olm-treated group were significantly lower than in the vehicle-treated group. Olm or RNH6270 reduced myeloperoxidase staining in the infarcted myocardium, decreased apoptosis in cultured cardiomyocytes and fibroblasts, as assessed by Hoechst staining and TUNEL assay, attenuated the accumulation of p53 and phosphorylated p53 and cleaved caspase 3 induced by MI or Ang II, as assessed by Western blotting, and up-regulated growth differentiation factor-15 (GDF-15). In cultured cardiomyocytes and fibroblasts, treatment with Ang II, Aldo or anoxia significantly down-regulated the expression of GDF-15.

CONCLUSIONS

Olm prevents cardiac rupture through inhibition of apoptosis and inflammation, which is attributable to the down-regulation of p53 activity and up-regulation of GDF-15. Our findings suggest that early administration of an AT1 receptor anatagonist to patients with acute MI is a potential preventive approach for cardiac rupture.

The gut-derived hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>/19 (FGF<em>15</em>/19) is an emerging versatile regulator of various metabolic pathways. As such, FGF<em>15</em>/19 has been implicated in homeostatic control of bile acid, carbohydrate and lipid metabolism in multiple target organs including the liver, adipose tissue and brain. In line with this, <em>growing</em> evidence suggests that dysregulation of FGF<em>15</em>/19 contributes to a number of metabolic and bile acid-associated disorders such as fatty liver disease, Type 2 diabetes and different gastrointestinal dysfunctions. In this review we summarize the current knowledge on the organ-specific functions of FGF<em>15</em>/19 and address their underlying molecular mechanisms. Moreover, recent advances in the characterization of <em>factors</em> that control the release of the hormone in the gut will be discussed and linked to the current view of how alterations of FGF<em>15</em>/19 signaling may contribute to disease development. Finally, the suitability of FGF<em>15</em>/19 as a potential therapeutic target will be critically reviewed.

The interaction between breast tumor epithelial and stromal cells is vital for initial and recurrent tumor <em>growth</em>. While breast cancer-associated stromal cells provide a favorable environment for proliferation and metastasis, the molecular mechanisms contributing to this process are not fully understood. Nuclear receptors (NRs) are intracellular transcription <em>factors</em> that directly regulate gene expression. Little is known about the status of NRs in cancer-associated stroma. Nuclear Receptor Low-Density Taqman Arrays were used to compare the gene expression profiles of all 48 NR family members in a collection of primary cultured cancer-associated <em>fibroblasts</em> (CAFs) obtained from estrogen receptor (ER)α positive breast cancers (n = 9) and normal breast adipose <em>fibroblasts</em> (NAFs) (n = 7). Thirty-three of 48 NRs were expressed in both the groups, while 11 NRs were not detected in either. Three NRs (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1); estrogen-related receptor beta (ERR-β); and RAR-related orphan receptor beta (ROR-β)) were only detected in NAFs, while one NR (liver receptor homolog-1 (LRH-1)) was unique to CAFs. Of the NRs co-expressed, four were significantly down-regulated in CAFs compared with NAFs (RAR-related orphan receptor-α (ROR-α); Thyroid hormone receptor-β (TR-β); vitamin D receptor (VDR); and peroxisome proliferator-activated receptor-γ (PPAR-γ)). Quantitative immunohistochemistry for LRH-1, TR-β, and PPAR-γ proteins in stromal <em>fibroblasts</em> from an independent panel of breast cancers (ER-positive (n = <em>15</em>), ER-negative (n = <em>15</em>), normal (n = 14)) positively correlated with mRNA expression profiles. The differentially expressed NRs identified in tumor stroma are key mediators in aromatase regulation and subsequent estrogen production. Our findings reveal a distinct pattern of NR expression that therefore fits with a sustained and increased local estrogen microenvironment in ER-positive tumors. NRs in CAFs may provide a new avenue for the development of intratumoral-targeted therapies in breast cancer.

Vasohibin‑2 (VASH2) is an angiogenic <em>factor</em>, and has been previously reported to be a cancer‑related gene, with cytoplasmic and karyotypic forms. In the current study VASH2 expression in human breast cancer tissue and adjacent non‑cancerous tissue was investigated with immunohistochemistry. MCF‑7 and BT474 human breast cancer cells were transfected with lentiviral constructs to generate in vitro VASH2 overexpression and knockdown models. In addition, BALB/cA nude mice were inoculated subcutaneously with transfected cells to generate in vivo models of VASH2 overexpression and knockdown. The effect of VASH2 on cell proliferation was investigated using a bromodeoxyuridine assay in vitro and immunohistochemistry of Ki67 in xenograft tumors. <em>Growth</em> <em>factors</em> were investigated using a human <em>growth</em> <em>factor</em> array, and certain <em>factors</em> were further confirmed by an immunoblot. The results indicated that the expression level of cytoplasmic VASH2 was higher in breast cancer tissues with a Ki67 (a proliferation marker) level of ≥14%, compared with tissues with a Ki67 level of <14%. VASH2 induced proliferation in vitro and in vivo. Four <em>growth</em> <em>factors</em> activated by VASH2 were identified as follows: <em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2), <em>growth</em>/differentiation <em>factor</em>‑<em>15</em> (GDF<em>15</em>), insulin‑like <em>growth</em> <em>factor</em>‑binding protein (IGFBP)3 and IGFBP6. FGF2 and GDF<em>15</em> may contribute to VASH2‑induced proliferation. The current study identified a novel role for VASH2 in human breast cancer, and this knowledge suggests that VASH2 may be a novel target in breast cancer treatment.

Healing of large open dermal wounds is associated with decreased values of the tensile strength even up to 6 months post-wounding. Results of previous studies have shown that healing is facilitated in the presence of a type I collagen sponge by promoting deposition of newly synthesized large-diameter collagen fibers parallel to the fibers of the sponge. In this study healing is evaluated in dermal wounds treated with a collagen sponge seeded with <em>fibroblasts</em> or coated with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Experimental results indicate that the presence of a collagen sponge results in increased wound tensile strength and increased collagen fiber diameters in the upper dermis <em>15</em> days post-wounding in an excisional guinea pig dermal wound model. In comparison, dermal wounds treated with collagen sponges seeded with <em>fibroblasts</em> or coated with bFGF showed increased tensile strengths <em>15</em> days postimplantation and increased degree of reepithelialization. These results indicate that <em>fibroblast</em> seeding and bFGF coating in conjunction with a type I collagen sponge matrix facilitate early dermal and epidermal wound healing.

BACKGROUND

Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells.

RESULTS

In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC) or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH6). The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF)3K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4 degrees C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes), and did not require localization of the enzyme to the nucleus.

CONCLUSIONS

The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells.

BACKGROUND

Flavopiridol is a flavonoid with antiproliferative effects mediated, in part, by inhibition of cyclin-dependent kinases. Clinical manifestations in a previous Phase I trial in patients with refractory malignancies treated with a 72-h flavopiridol infusion included a proinflammatory syndrome consisting of fever, fatigue, and "local" tumor pain with concomitant alterations in plasma acute-phase reactant proteins.

OBJECTIVE

The aim of this study was to determine whether the proinflammatory syndrome observed in this trial was associated with modulation of plasma cytokines.

METHODS

Patients receiving flavopiridol (n = 76) had serial plasma samples drawn preinfusion and during the infusion for evaluation of interleukin (IL)-6, IL-10, IL-12, granulocyte macrophage colony-stimulating factor, basic-fibroblast growth factor, transforming growth factor-beta, and tumor necrosis factor-alpha levels by standard ELISA assays. The Wilcoxon signed rank test was used to test the significance of the difference between the baseline (time 0) plasma cytokine levels compared with the values of each subsequent data collection time points (8, 24, 48, and 72 h).

RESULTS

There was a significant and sustained increase in plasma IL-6 levels at all time points when compared with baseline values. Paired values were used in the statistical analysis. Median plasma (interquartile range) values of IL-6 were elevated from 15.5 (9-52) pg/ml at baseline to 23 (4-48) pg/ml (P < 0.01) at 8 h; from 15 (2-48) pg/ml at baseline to 46 (21-105) pg/ml (P < 0.001) at 24 h; from 16 (9-52) pg/ml at baseline to 61 (32-170) pg/ml (P < 0.001) at 48 h; and from 15.5 (6-48) pg/ml to 68 (40-200) pg/ml (P < 0.001) at 72 h. Significance was maintained even when adjusted for multiple comparisons. The relative increase in IL-6 concentration was dose-dependent. Moreover, IL-6 elevation had a direct correlation with flavopiridol peak plasma concentration, flavopiridol area under the curve, and plasma C-Reactive protein levels. A significant decrease in plasma granulocyte macrophage colony-stimulating factor occurred at the 8-h sampling point: 50 pg/ml (interquartile range 10-205 pg/ml, P < 0.01) when compared with baseline plasma levels and 71 pg/ml (interquartile range 5-152 pg/ml, P < 0.01). No changes in the other pro or anti-inflammatory cytokines were observed. Immunohistochemistry studies in bone marrow aspirates from a prospective group of patients in this trial demonstrated approximately 4-fold induction of IL-6 (compared with baseline), mostly in non-T cells.

CONCLUSIONS

Biochemical analysis of plasma in patients undergoing infusional flavopiridol found a significant dose-dependent induction of IL-6. IL-6 elevation could be a marker for the process leading to the appearance of the proinflammatory syndrome observed in patients treated with infusional flavopiridol. The mechanism(s) underlying IL-6 induction and its significance are still unknown but may influence strategies to modulate flavopiridol's clinical effects.

BACKGROUND

Phosphorus-based food additives can substantially increase total phosphorus intake per day, but the effect of these additives on endocrine factors regulating bone and mineral metabolism is unclear.

OBJECTIVE

This study aimed to examine the effect of phosphorus additives on markers of bone and mineral metabolism. Design and Setting, and Participants: This was a feeding study of 10 healthy individuals fed a diet providing ∼1000 mg of phosphorus/d using foods known to be free of phosphorus additives for 1 week (low-additive diet), immediately followed by a diet containing identical food items; however, the foods contained phosphorus additives (additive-enhanced diet). Parallel studies were conducted in animals fed low- (0.2%) and high- (1.8%) phosphorus diets for 5 or 15 weeks.

METHODS

The changes in markers of mineral metabolism after each diet period were measured.

RESULTS

Participants were 32 ± 8 years old, 30% male, and 70% black. The measured phosphorus content of the additive-enhanced diet was 606 ± 125 mg higher than the low-additive diet (P < .001). After 1 week of the low-additive diet, consuming the additive-enhanced diet for 1 week significantly increased circulating fibroblast growth factor 23 (FGF23), osteopontin, and osteocalcin concentrations by 23, 10, and 11%, respectively, and decreased mean sclerostin concentrations (P < .05 for all). Similarly, high-phosphorus diets in mice significantly increased blood FGF23, osteopontin and osteocalcin, lowered sclerostin, and decreased bone mineral density (P < .05 for all).

CONCLUSIONS

The enhanced phosphorus content of processed foods can disturb bone and mineral metabolism in humans. The results of the animal studies suggest that this may compromise bone health.

Tissue engineering has revealed the potential to regenerate injured vocal folds, and identification of the most suitable seed cells has remained a hot topic of research. The aim of this study was to implant <em>fibroblast</em>-like cells differentiated from adipose-derived mesenchymal stem cells (ADSCs) in a canine acute vocal fold wound model. We then sought to characterize changes in the extracellular matrix (ECM) proteins of vocal fold lamina propria. For this purpose, ADSCs were induced to differentiate into <em>fibroblasts</em> under the regulation of connective tissue <em>growth</em> <em>factor</em> in vitro. Cell surface proteins were identified by immunofluorescence staining. Thirty vocal folds of 17 canines were injured by localized resection and injected with <em>fibroblast</em>-like cells (differentiated ADSCs, dADSCs), ADSCs or vocal fold <em>fibroblasts</em> (VFFs). The morphology of vocal folds was observed, and the characteristics of ECM protein components (collagen, elastin, hyaluronic acid, decorin and fibronectin) were evaluated by immunofluorescence staining from <em>15</em> days to 6 months following implantation. The results showed that in vitro, the dADSCs showed morphology and cell surface protein expression similar to those of VFFs. After implantation in vivo, the surfaces of the recipient vocal folds became almost smooth in the dADSCs and ADSCs groups at 6 months but remained slightly concave and stiff in the VFFs group. The elastin fluorescence intensity increased significantly and was maintained at a high level in the dADSCs group. The collagen fluorescence intensity increased slightly in the dADSCs and ADSCs groups, whereas it demonstrated a more irregular arrangement in the VFFs group. The fluorescence intensity of hyaluronic acid, decorin and fibronectin was similar between the three implanted groups. These results indicated that dADSCs may confer an advantage for vocal fold wound healing. Furthermore, dADSCs have the ability to secrete ECM components in vivo, particularly elastin, which may be beneficial for vocal fold vibration recovery.

We sought to identify novel urinary biomarkers of kidney function in type 2 diabetes. We screened the renal transcriptome of db/db and db/m mice for differentially expressed mRNA transcripts that encode secreted proteins with human orthologs. Whether elevated urine levels of the orthologous proteins correlated with diminished glomerular filtration rate was tested in a cross-sectional study of n = 56 patients with type 2 diabetes. We identified 36 putative biomarker genes in db/db kidneys: 31 upregulated and 5 downregulated. Urinary protein levels of six selected candidates (endothelin-1, lipocalin-2, transforming <em>growth</em> <em>factor</em>-β, <em>growth</em> and differentiation <em>factor</em>-<em>15</em>, interleukin-6, and macrophage chemoattractant protein-1) were elevated in type 2 diabetic patients with subnormal glomerular filtration rate (i.e., <90 ml·min(-1)·1.73 m(-2)), independent of microalbuminuria, age, sex, race, and use of angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists. In contrast, urinary levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> were not increased. A composite variable of urine albumin and any of the six candidate markers was associated with subnormal estimated glomerular filtration rate more closely than albumin alone. In addition, urinary endothelin-1, <em>growth</em> and differentiation <em>factor</em>-<em>15</em>, and interleukin-6 were associated with a marker of proximal tubule damage, N-acetyl-β-d-glucosaminidase activity. These results suggest that gene expression profiling in diabetic mouse kidney can complement existing proteomic-based approaches for renal biomarker discovery in humans.

Produced by senescent cells, the senescence-associated secretory phenotype (SASP) is a potential driver of age-related dysfunction. We tested whether circulating concentrations of SASP proteins reflect age and medical risk in humans. We first screened senescent endothelial cells, <em>fibroblasts</em>, preadipocytes, epithelial cells, and myoblasts to identify candidates for human profiling. We then tested associations between circulating SASP proteins and clinical data from individuals throughout the life span and older adults undergoing surgery for prevalent but distinct age-related diseases. A community-based sample of people aged 20-90 years (retrospective cross-sectional) was studied to test associations between circulating SASP <em>factors</em> and chronological age. A subset of this cohort aged 60-90 years and separate cohorts of older adults undergoing surgery for severe aortic stenosis (prospective longitudinal) or ovarian cancer (prospective case-control) were studied to assess relationships between circulating concentrations of SASP proteins and biological age (determined by the accumulation of age-related health deficits) and/or postsurgical outcomes. We showed that SASP proteins were positively associated with age, frailty, and adverse postsurgery outcomes. A panel of 7 SASP <em>factors</em> composed of <em>growth</em> differentiation <em>factor</em> <em>15</em> (GDF<em>15</em>), TNF receptor superfamily member 6 (FAS), osteopontin (OPN), TNF receptor 1 (TNFR1), ACTIVIN A, chemokine (C-C motif) ligand 3 (CCL3), and IL-<em>15</em> predicted adverse events markedly better than a single SASP protein or age. Our findings suggest that the circulating SASP may serve as a clinically useful candidate biomarker of age-related health and a powerful tool for interventional human studies.

Keywords: Aging; Cellular senescence.

Apart from several established clinical risk factors for atrial fibrillation (AF), a number of biomarkers have also been identified as potential risk factors for AF. None of these have so far been adopted in clinical practice.

To use a novel custom-made proteomics chip to discover new prognostic biomarkers for AF risk.

In two independent community-based cohorts (Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study (978 participants without AF, mean age 70.1 years, 50% women, median follow-up 10.0 years) and Uppsala Longitudinal Study of Adult Men (ULSAM) (n=725, mean age 77.5 years, median follow-up 7.9 years)), ninety-two plasma proteins were assessed at baseline by a proximity extension assay (PEA) chip. Of those, 85 proteins showed a call rate >70% in both cohorts.

Thirteen proteins were related to incident AF in PIVUS (148 events) using a false discovery rate of 5%. Of those, five were replicated in ULSAM at nominal multivariable p value (123 events, N-terminal pro-B-type natriuretic peptide (NT-pro-BNP), fibroblast growth factor 23 (FGF-23), fatty acid-binding protein 4 (FABP4), growth differentiation factor 15 (GDF-15) and interleukin-6 (IL-6)). Of those, NT-pro-BNP and FGF-23 were also associated with AF after adjusting for established AF risk factors. In a prespecified secondary analysis pooling the two data sets, T-cell immunoglobulin and mucin domain 1 (TIM-1) and adrenomedullin (AM) were also significantly related to incident AF in addition to the aforementioned five proteins (Bonferroni-adjustment). The addition of NT-pro-BNP to a model with established risk factors increased the C-statistic from 0.605 to 0.676 (p<0.0001).

Using a novel proteomics approach, we confirmed the previously reported association between NT-pro-BNP, FGF-23, GDF-15 and incident AF, and also discovered four proteins (FABP4, IL-6, TIM-1 and AM) that could be of importance in the development of AF.

BACKGROUND

The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells.

RESULTS

We used mouse ES cells stably transfected with a cardiac-specific α-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time- and concentration-dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial co-administration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation.

CONCLUSIONS

FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications.

Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is present in vivo and in vitro. In vivo, tear samples were collected from age-matched controls with no eye disease (n = 36) and KC diagnosed subjects (n = 17). Samples were processed for proteomics using LC-MS/MS. In vitro, cells were isolated from controls (Human Corneal <em>Fibroblasts</em>-HCF) and KC subjects (Human Keratoconus Cells-HKC) and stimulated with a Vitamin C (VitC) derivative for 4 weeks, and with one of the three transforming <em>growth</em> <em>factor</em>-beta (TGF-β) isoforms. Samples were analyzed using real-time PCR and Western Blots. By using proteomics analysis, the Gross cystic disease fluid protein-<em>15</em> (GCDFP-<em>15</em>) or prolactin-inducible protein (PIP) was found to be the best independent biomarker able to discriminate between KC and controls. The intensity of GCDFP-<em>15</em>/PIP was significantly higher in healthy subjects compared to KC-diagnosed. Similar findings were seen in vitro, using a 3D culture model. All three TGF-β isoforms significantly down-regulated the expression of GCDFP-<em>15</em>/PIP. Zinc-alpha-2-glycoprotein (AZGP1), a protein that binds to PIP, was identified by proteomics and cell culture to be highly regulated. In this study by different complementary techniques we confirmed the potential role of GCDFP-<em>15</em>/PIP as a novel biomarker for KC disease. It is likely that exploring the GCDFP-<em>15</em>/PIP-AZGP1 interactions will help better understand the mechanism of KC disease.

The addition of platelet-derived <em>growth</em> <em>factor</em> (PDGF) to serum-starved <em>fibroblasts</em> induces increased motility, formation of lamellipodia, increased ruffling activity, and actin ring structures associated with dorsal ruffles. Involvement of the phosphatidylinositol cycle (PI-cycle) in these morphological changes was investigated by observing the effects of neomycin, an inhibitor of the PI-cycle, on cultured human foreskin <em>fibroblasts</em>. The role of actin in the changes was investigated by using cytochalasin D (CD). Actin in detergent-extracted cells was labelled with TRITC-phalloidin and examined with fluorescence microscopy. Using PDGF and neomycin simultaneously potentiated lamellipodia formation, ruffling activity, as well as the number of cells with actin rings. Furthermore, neomycin by itself induced morphological changes similar to those induced by PDGF. Quantitation of actin rings showed dose and time dependency for PDGF and neomycin respectively, with a maximal number of cells containing rings after <em>15</em> min of exposure to either 3.5 mM neomycin or 10 ng PDGF/ml. Comparing the two substances, PDGF induced ring formation in a greater number of cells. These processes were inhibited by the presence of CD. PDGF- and neomycin-induced changes in the actin cytoskeleton were also observed in human embryonic lung <em>fibroblasts</em>, human glial cells, and embryonic mouse <em>fibroblasts</em>, all of which are known to express PDGF-receptors. In conclusion, the present study indicates that an increased turnover of the PI-cycle is not essential for the changes in actin organization induced by PDGF.

OBJECTIVE

Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA).

METHODS

Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict "effective" and "ineffective" triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry.

RESULTS

Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%-80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability.

CONCLUSIONS

Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2x L-<em>15</em> media. Various supplements including <em>growth</em> <em>factors</em>, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with large nuclei) were maintained for up to 17 months. Epithelioid cells grew best in modified Grace's medium but were generally short-lived (less than two months). <em>Fibroblast</em>-like cells formed confluent monolayers in modified 2x L-<em>15</em> medium, were passaged three times and survived for 17 months. In other cultures, millions of rounded cells migrated from tissue. They survived for prolonged periods (up to ten months), either loosely attached to the flask or suspended in the medium. A change in dominant cell type from fibroblastic to epithelioid was observed in some cultures after three or nine months incubation. These epithelioid cells which had very large nuclei, grew to confluence but could not be sub-cultured. It is noteworthy that the rounded cells and the epithelioid cells with the large nuclei both produced vitellogenin in protein-free media.

In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic <em>fibroblasts</em> (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for <em>15</em> passages (about 100 days). Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous <em>growth</em> <em>factors</em>. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like <em>growth</em> <em>factor</em> 2 (IGF-2), and transforming <em>growth</em> <em>factor</em> β (TGF-β), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.