Human acute myeloid leukemia (AML) cells, like normal hematopoietic progenitors, die rapidly by apoptosis when cultured under serum-free conditions. Apoptosis was demonstrated by electron microscopy and agarose gel electrophoresis and quantified by flow cytometry. Culturing AML blasts in the presence of a bone marrow <em>fibroblast</em> (BMF) monolayer reduced the percentage of AML blasts undergoing apoptosis in the majority of cases studied. The effect was more pronounced when AML cells were cultured in the presence of an adherent long-term bone marrow (LTBM) stroma rather than BMF. Overall, the mean percentage of AML cells with fragmented DNA fell from 85 +/- 8% in control cultures to <em>20</em> +/- 9% in cultures with adherent stroma (p = 0.0004, n = 7). Supplementation of serum-free medium with recombinant cytokines, including stem cell <em>factor</em>, granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF), and tumor necrosis <em>factor</em> (TNF)-alpha or with human placenta-conditioned medium (HPCM) matched the degree of inhibition of apoptosis induced by BMF in only 50% of cases. Granulocyte colony-stimulating <em>factor</em> (G-CSF), interleukin-1 beta (IL-1 beta), and IL-6 were completely ineffective. Consistent with this observation, direct contact between leukemic cells and adherent layers was essential for maximum inhibition of leukemic-cell DNA fragmentation. Separation by a porous membrane allowing passage of soluble <em>growth</em> <em>factors</em>, but interrupting direct cell contact, was associated with significantly greater DNA fragmentation and cell death. Inhibition of leukemic-cell apoptosis correlated with improved survival and <em>growth</em> of malignant clonogenic cells. Colonies grown in cultures were identified as leukemic by morphology and by fluorescence in in situ hybridization to demonstrate numerical chromosomal abnormalities identified at diagnosis. Close contact between leukemic cells and bone marrow inhibits blast cell apoptosis and directly promotes survival of clonogenic AML cells.

Effective diabetic kidney disease (DKD) biomarkers remain elusive, and urinary miRNAs represent a potential source of novel noninvasive disease sentinels. We profiled 754 miRNAs in pooled urine samples from DKD patients (n = <em>20</em>), detecting significantly increased miR-126, miR-155, and miR-29b compared with controls (n = <em>20</em>). These results were confirmed in an independent cohort of 89 DKD patients, 62 diabetic patients without DKD, and 41 controls: miR-126 (2.8-fold increase; P < 0.0001), miR-155 (1.8-fold increase; P < 0.001), and miR-29b (4.6-fold increase; P = 0.024). Combined receiver operating characteristic curve analysis resulted in an area under the curve of 0.8. A relative quantification threshold equivalent to 80% sensitivity for each miRNA gave a positive signal for 48% of DKD patients compared with 3.6% of diabetic patients without DKD. Laser-capture microdissection of renal biopsy specimens, followed by quantitative RT-PCR, detected miR-155 in glomeruli and proximal and distal tubules, whereas miR-126 and miR-29b were most abundant in glomerular extracts. Subsequent experiments showed miR-126 and miR-29b enrichment in glomerular endothelial cells (GEnCs) compared with podocytes, proximal tubular epithelial cells, and <em>fibroblasts</em>. Significantly increased miR-126 and miR-29b were detected in GEnC conditioned medium in response to tumor necrosis <em>factor</em>-α and transforming <em>growth</em> <em>factor</em>-β1, respectively. Our data reveal an altered urinary miRNA profile associated with DKD and link these variations to miRNA release from GEnCs.

OBJECTIVE

The growth behaviour of well-differentiated neuroendocrine carcinomas of the gastro-entero-pancreatic system varies greatly and parameters predicting their prognosis are lacking. The aim of our study was to investigate whether tumour growth could be correlated with the release of proangiogenic factors into the circulation.

METHODS

Circulating vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF) and angiogenin were measured in 38 patients with advanced neuroendocrine carcinomas and compared to healthy age-matched controls. In 20 patients, angiogenic cytokine levels were measured at consecutive time points and correlated to tumour progression as assessed by abdominal CT scan, MRI and chromogranin A levels.

RESULTS

VEGF levels were elevated in patients compared to controls (P < 0.002) and clearly associated with tumour progression (P < 0.005). Angiogenin levels were significantly higher in patients than in controls (P < 0.003), while high IL-8 levels were predictive of shorter survival. Angiogenin and bFGF levels were correlated neither with tumour growth nor with patient survival.

CONCLUSIONS

VEGF and IL-8 are associated with tumour progression and might qualify as markers of prognosis and therapy control in patients with neuroendocrine carcinomas. Our results support the notion that specific anti-angiogenic therapies should be evaluated in neuroendocrine carcinoma patients.

OBJECTIVE

Do platelets play any role in the development of adenomyosis?

CONCLUSIONS

As in endometriosis, adenomyotic lesions show significantly increased platelet aggregation, increased expression of transforming growth factor (TGF)-β1, phosphorylated Smad3, markers of epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT), and smooth muscle metaplasia (SMM), in conjunction with increased fibrosis as compared with normal endometrium.

BACKGROUND

Both EMT and FMT are known to play vital roles in fibrogenesis in general and in endometriosis in particular. EMT has been implicated in the development of adenomyosis. SMM is universally seen in endometriosis and also in adenomyosis, and is correlated positively with the extent of fibrosis. However, there has been no published study on the role of platelets in fibrogenesis in adenomyosis, even though adenomyotic lesions undergo repeated cycles of tissue injury and repair, which suggests the involvement of platelets and their possible roles in fibrogenesis.

METHODS

Cross-sectional studies of ectopic endometrial and control endometrial tissue samples from three sets of women with and without adenomyosis (n= 34 and 20, 12 and 10, and 8 and 8, respectively) were carried out from 2014 to 2015.

METHODS

Immunohistochemistry analysis of ectopic endometrial tissues from women with (n= 34) and without (n= 20) adenomyosis with respect to biomarkers of EMT, FMT and highly differentiated smooth muscle cells as well as TGF-β1, phosphorylated Smad3, markers of proliferation, angiogenesis and extracellular matrix (ECM) deposits. Masson trichrome staining, Van Gieson staining and Pico-Sirius staining were performed to evaluate and quantify the extent of fibrosis in lesions. Progesterone receptor isoform B (PR-B) staining also was performed. In addition, CD42b-positive platelets in ectopic (n= 12) and control (n= 10) endometrium were counted by confocal microscopy and compared. The protein expression levels of TGF-β1 and phosphorylated Smad3 in both ectopic (n= 8) and control (n= 8) endometrium were measured by western blot analysis. Immunofluorescent staining of both platelets and hepatocyte growth factor (HGF) was also performed for adenomyotic tissue samples (n= 10).

RESULTS

Adenomyotic lesions had a significantly higher extent of platelet aggregation and increased staining for TGF-β1 and phosphorylated Smad3 (both P-values <0.001 versus control). In addition, E-cadherin staining was decreased while vimentin staining in adenomyotic epithelial cells was increased, along with increased staining of proliferating cell nuclear antigen, vascular endothelial growth factor and CD31 (all P-values <0.001), markers of proliferation and angiogenesis. Staining for α-SMA, a marker for myofibroblast, desmin, smooth muscle myosin heavy chain and oxytocin receptor was significantly increased in adenomyotic lesions versus control, concomitant with increased staining of collagen I and lysyl oxidase (all P-values <0.001). Histochemistry analysis indicates that the extent of fibrosis is high in adenomyotic lesions (P < 0.001), and the extent appeared to correlate negatively with the microvessel density (P < 0.05). PR-B staining was significantly decreased in adenomyotic lesion as compared with control endometrium (P < 0.001). Platelets and HGF were co-localized mostly in the stromal component of adenomyotic lesions, near the glandular epithelium.

CONCLUSIONS

The results are limited by the cross-sectional nature of the study and the use of histochemistry and immunohistochemistry analyses only, but nonetheless is a validation of our previous finding in mouse experiments.

CONCLUSIONS

The data presented are consistent with the notion that platelet-induced activation of the TGF-β/Smad signaling pathway may be a driving force in EMT, FMT and SMM in the development of adenomyosis, leading to fibrosis. This study provides the first piece of evidence that adenomyotic lesions are wounds that undergo repeated injury and healing, and, as such, platelets play critical roles in the development of adenomyosis by promoting proliferation, angiogenesis, increasing ECM deposits, and SMM, resulting in fibrosis. Platelets may also be involved in uterine hyperactivity and myometrial hyperinnervation. Our results provide one explanation as to why adenomyosis is a challenge for medical treatment, and shed new light onto the pathophysiology of adenomyosis.

BACKGROUND

Support for data collection and analysis was provided by grants from the National Science Foundation of China. None of the authors has anything to disclose.

BACKGROUND

Recent studies have linked histone deacetylases (HDAC) to remodeling of the heart and cardiac fibrosis in heart failure. However, the molecular mechanisms linking chromatin remodeling events with observed anti-fibrotic effects are unknown. Here, we investigated the molecular players involved in anti-fibrotic effects of HDAC inhibition in congestive heart failure (CHF) myocardium and cardiac fibroblasts in vivo.

RESULTS

MI was created by coronary artery occlusion. Class I HDACs were inhibited in three-week post MI rats by intraperitoneal injection of Mocetinostat (20 mg/kg/day) for duration of three weeks. Cardiac function and heart tissue were analyzed at six week post-MI. CD90+ cardiac fibroblasts were isolated from ventricles through enzymatic digestion of heart. In vivo treatment of CHF animals with Mocetinostat reduced CHF-dependent up-regulation of HDAC1 and HDAC2 in CHF myocardium, improved cardiac function and decreased scar size and total collagen amount. Moreover, expression of pro-fibrotic markers, collagen-1, fibronectin and Connective Tissue Growth Factor (CTGF) were reduced in the left ventricle (LV) of Mocetinostat-treated CHF hearts. Cardiac fibroblasts isolated from Mocetinostat-treated CHF ventricles showed a decrease in expression of collagen I and III, fibronectin and Timp1. In addition, Mocetinostat attenuated CHF-induced elevation of IL-6 levels in CHF myocardium and cardiac fibroblasts. In parallel, levels of pSTAT3 were reduced via Mocetinostat in CHF myocardium.

CONCLUSIONS

Anti-fibrotic effects of Mocetinostat in CHF are associated with the IL-6/STAT3 signaling pathway. In addition, our study demonstrates in vivo regulation of cardiac fibroblasts via HDAC inhibition.

Fibroblast growth factor 19 (FGF19) and 21 (FGF21) have emerged as key regulators of energy homeostasis. Our aim was to analyze the impact of weight loss (WL) induced either by conventional dietary treatment (CDT) or bariatric surgery on FGF19 and FGF21 concentrations. Furthermore, the diverse effect of sleeve gastrectomy (SG) versus RYGB (Roux-en-Y gastric bypass) as two surgical procedures that affect the gastrointestinal anatomy and physiology differently was also analyzed.

Serum concentrations of FGF19 and FGF21 were measured in 137 obese patients with different degrees of insulin resistance matched by sex, age and body adiposity and compared to 33 lean individuals. Furthermore, FGF19 and FGF21 were measured in 114 subjects before and one-year after WL induced either by CDT (n = 28), SG (n = 20) or RYGB (n = 66).

Circulating serum FGF19 concentrations were decreased (P < 0.01) similarly in obese patients regardless of their degree of insulin resistance, while FGF21 levels were increased in obesity (P < 0.01), being further increased in obesity-associated T2D (P < 0.01). FGF19 concentrations were increased in obese subjects after surgically-induced WL (P < 0.01), but not after WL achieved by CDT, while FGF21 levels were reduced after CDT- (P < 0.05) or SG-induced WL (P < 0.05), but not after RYGB. The change in FGF21 concentrations emerged as a significant predictor of the change in insulin resistance (HOMA) after WL.

Based on the circulating concentrations and their subsequent pattern of response following WL, we conclude that FGF19 levels are mainly related to body adiposity, in particular visceral adiposity, while FGF21 is mainly related to glucose homeostasis. CLINICALTRIALS.

NCT01572090.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (FGF<em>20</em>) is a neurotrophic <em>factor</em> preferentially expressed in the substantia nigra of rat brain and could be involved in dopaminergic neurons survival. Recently, a strong genetic association has been found between FGF<em>20</em> gene and the risk of suffering from Parkinson's disease (PD). Our aim was to replicate this association in two independent populations.

METHODS

Allelic, genotypic, and haplotype frequencies of four biallelic polymorphisms were assessed in 151 sporadic PD cases and 186 controls from Greece, and 144 sporadic PD patients and 135 controls from Finland.

RESULTS

No association was found in any of the populations studied.

CONCLUSIONS

Taken together, these findings suggest that common genetic variants in FGF<em>20</em> are not a risk <em>factor</em> for PD in, at least, some European populations.

Preoligodendrocytes have been described in cultures and tissue prints of adult human white matter (Armstrong et al., 1992). To characterize further these precursors of human oligodendrocytes, we have investigated whether they express genes playing a critical role in oligodendrocyte development. In the intact human brain, platelet-derived <em>growth</em> <em>factor</em> receptor alpha (PDGF alpha R) and myelin transcription <em>factor</em> 1 (MyTI) transcripts are expressed in 1-2% of cells of the oligodendrocyte lineage (OL), and clusters of such cells can be found in the periventricular region. Myelin basic protein transcripts containing exon 2 information (exon 2+ MBP), which are characteristic of the premyelinating stage, are detected in 15-<em>20</em>% of OL cells in vivo. When OL cells are separated from human white matter and allowed to regenerate in vitro, a much larger proportion of these cells express developmentally regulated genes, while exon 2- MBP and proteolipid protein (PLP) transcripts characteristic of mature OL cells appear transiently downregulated. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), even in the presence of PDGF, does not promote DNA synthesis in these cultured OL cells. Yet bFGF induces human oligodendrocytes to regenerate their processes rapidly in vitro and to express O4 antigens as well as exon 2+ MBP, MyTI, and PLP transcripts. While bFGF accelerates early regenerative processes, it also maintains high expression of exon 2+ MBP transcripts in OL cells for up to 2 weeks in vitro. In contrast, high levels of insulin in the absence of bFGF allow accumulation of exon 2- MBP and PLP transcripts in most OL cells at 2-3 weeks in vitro. We propose that the myelinated human brain harbors a small pool of precursors of oligodendrocytes and that <em>growth</em> <em>factor</em>-regulated phenotypic plasticity rather than mitogenic potential accounts for the regeneration of oligodendrocytes in the initial stages of demyelinating diseases such as multiple sclerosis.

Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and some members of the transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) superfamily, including TGF-betas themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF-beta superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein-1 (OP-1; BMP-7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP-1 at both ortho- and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF-beta1. The single application of relatively high doses of TGF-beta1 (<em>20</em> ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP-1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP-1) dose-dependent angiogenic response. The binary application of a relatively low dose of OP-1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (<em>20</em> ng) dose of TGF-beta1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective <em>factors</em>. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis.

XFE progeroid syndrome, a disease of accelerated aging caused by deficiency in the DNA repair endonuclease XPF-ERCC1, is modeled by Ercc1 knockout and hypomorphic mice. Tissues and primary cells from these mice senesce prematurely, offering a unique opportunity to identify <em>factors</em> that regulate senescence and aging. We compared microRNA (miRNA) expression in Ercc1-/- primary mouse embryonic <em>fibroblasts</em> (MEFs) and wild-type (WT) MEFs in different <em>growth</em> conditions to identify miRNAs that drive cellular senescence. Microarray analysis showed three differentially expressed miRNAs in passage 7 (P7) Ercc1-/- MEFs grown at <em>20</em>% O2 compared to Ercc1-/- MEFs grown at 3% O2. Thirty-six differentially expressed miRNAs were identified in Ercc1-/- MEFs at P7 compared to early passage (P3) in 3% O2. Eight of these miRNAs (miR-449a, miR-455*, miR-128, miR-497, miR-543, miR-450b-3p, miR-872 and miR-10b) were similarly downregulated in the liver of progeroid Ercc1-/Δ and old WT mice compared to adult WT mice, a tissue that senesces with aging. Three miRNAs (miR-449a, miR-455* and miR-128) were also downregulated in Ercc1-/Δ and WT old mice kidneys compared to young WT mice. We also discovered that the miRNA expression regulator Dicer is significantly downregulated in tissues of old mice and late passage cells compared to young controls. Collectively these results support the conclusion that the miRNAs identified may play an important role in staving off cellular senescence and their altered expression could be indicative of aging.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) are two <em>growth</em> <em>factors</em> that will modulate chondrocyte <em>growth</em> and matrix synthesis. Here we report that these two <em>growth</em> <em>factors</em> act in a synergistic fashion to suppress the synthesis of type II collagen by embryonic chicken sternal chondrocytes. Treatment of chondrocytes with <em>20</em> ng/ml TGF-beta or 100 ng/ml FGF (acidic or basic) results in a 60-70% suppression of expression of the pro alpha 1 chain of type II collagen. By comparison, when chondrocytes are exposed to a combination of 1 ng/ml TGF-beta and 10 ng/ml FGF, a complete suppression of type II collagen synthesis was observed. Epidermal <em>growth</em> <em>factor</em> (EGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), or insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1) produce no suppression of synthesis either individually or in combination with TGF-beta. The decreased expression of the protein results from a decrease in the steady-state level of the mRNA transcript coding for type II procollagen, as indicated by a northern analysis. Finally, chondrocytes transfected with a plasmid carrying the CAT gene driven by the collagen II promoter/enhancer sequence displayed high levels of CAT activity when cultured in control media, but treatment of the cells with a combination of the two <em>growth</em> <em>factors</em> resulted in a dramatic reduction of CAT activity, indicating diminished promoter activity. These results suggest that both TGF-beta and FGF can down-regulate transcription of the collagen II gene through regulatory DNA sequences in the promoter and/or enhancer region. In addition, the finding of synergy suggests that these two <em>growth</em> <em>factors</em> may act through different pathways.

BACKGROUND

To correlate serum cytokine and angiogenic factor (CAF) levels with overall survival (OS) in metastatic renal cell carcinoma (mRCC) treated with interferon-alpha (IFN-alpha).

METHODS

Serum CAF levels were measured in 103 patients treated on a randomized trial with IFN-alpha 0.5 million units (MU) twice daily or 5 MU daily. Concentrations of 17 analytes were determined by multiplex bead immunoassays [vascular endothelial growth factor A (VEGF(A)) and several cytokines] or enzyme-linked immunosorbent assay (basic fibroblast growth factor). We used proportional hazards models to evaluate the effect of CAF levels and clinical factors on OS.

RESULTS

Pretreatment serum interleukin (IL) 5, IL-12 p40, VEGF(A), and IL-6 levels and Memorial Sloan-Kettering Cancer Center risk grouping independently correlated with OS, with hazard ratios of 2.33, 2.00, 2.07, 1.82, and 0.39, respectively (concordance index = 0.69 for the combined model versus 0.60 for the CAF model versus 0.52 for the clinical model). Based on an index derived from these five risk factors (RFs), patients with 0-2 RF had a median OS time of 32 months versus 9 months for patients with 3-5 RF (P < 0.0001).

CONCLUSIONS

Serum CAF profiling contributes to prognostic evaluation in mRCC and helps to identify a subset of patients with 20% 5-year OS.

BACKGROUND

Circadian rhythms in plasma concentrations of many hormones and cytokines determine their effects on target cells.

METHODS

Circadian variations were studied in cortisol, melatonin, cytokines (basic fibroblast growth factor IbFGF], EGF, insulin-like growth factor-1 [IGF-1]), and a cytokine receptor (insulin-like growth factor binding protein-3 [IGFBP-3]) in the plasma of 28 patients with metastatic breast cancer. All patients followed a diurnal activity pattern. Blood was drawn at 3h intervals during waking hours and once during the night, at 03:00. The plasma levels obtained by enzyme-linked immunoassay (ELISA) or radioimmunoassay (RIA) were evaluated by population mean cosinor (using local midnight as the phase reference) and by one-way analysis of variance (ANOVA).

RESULTS

Cortisol and melatonin showed a high-amplitude circadian rhythm and a superimposed 12h frequency. bFGF showed a circadian rhythm with an acrophase around 13:00 with a peak-to-trough interval (double amplitude) of 18.2% and a superimposed 12h frequency. EGF showed a circadian rhythm with an acrophase around 14:20, a peak-to-trough interval of 25.8%, and a superimposed 12h frequency. IGF-1 showed a high value in the morning, which is statistically different (t test) from the low value at 10:00, but a regular circadian or ultradian rhythm was not recognizable as a group phenomenon. IGFBP-3 showed a low-amplitude (peak-to-trough difference 8.4%) circadian rhythm with the acrophase around 11:00 and low values during the night.

CONCLUSIONS

(1) Circadian periodicity is maintained in hospitalized patients with metastatic breast cancer. (2) Ultradian (12h) variations were superimposed on the circadian rhythms of the hormones and several of the cytokines measured. (3) Studies of hormones and cytokines in cancer patients have to take their biologic rhythms into consideration. (4) The circadian periodicity of tumor growth stimulating or restraining factors raises questions about circadian and/or ultradian variations in the pathophysiology of breast cancer.

The use of <em>growth</em> <em>factors</em> for the therapeutic stimulation of neovascularization in regenerative medicine has been extensively investigated, but the inability to control their temporal delivery may limit clinical success. A strategy that delivers continuous therapeutic concentrations of <em>growth</em> <em>factors</em> may increase the protein's efficacy. The present study investigates the ability of sustained delivery of <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1), to induce neovascularization in vivo. Alginate microbeads were synthesized to release active FGF-1 for three weeks. Microbeads loaded with FGF-1 (total amount 150 ng) were implanted into a surgically created omentum pouch in rats and were compared to control empty microbead implants and a single bolus injection of 150 ng of FGF-1 with empty microbead implant. Animals were sacrificed at either 3 or 6 weeks post implantation and omenta were analyzed for vascular density and mural cell interactions. Vascular area for bolus FGF-1 and FGF-1 loaded microbeads was higher than control at 3 weeks. At 6 weeks, vascular density in the group with FGF-1 loaded microbeads was significantly higher than the group with bolus administration of FGF-1, primarily due to an increase in the number of vessels less than <em>20</em> microm in diameter. Vascular density in omenta of the group receiving the bolus FGF-1 returned to control levels by 6 weeks. Staining for smooth muscle actin showed that 50% of vessels had associated mural cells. There was a trend of increased mural cell staining at 6 weeks for the FGF-1 loaded beads compared to bolus FGF-1 and control levels. Results in these studies suggest that sustained release of FGF-1 increases the duration of the vascular response in contrast to a bolus injection of FGF-1.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) confers acute, preconditioning-like cardiac resistance to ischemic injury in a protein kinase C (PKC)-dependent fashion. One of the downstream targets of PKC is the gap junction protein connexin-43 (Cx43). We thus examined the effects of FGF-2 on Cx43 phosphorylation at specific PKC sites in the adult heart. Rat hearts perfused ex vivo for <em>20</em> min with an FGF-2-containing solution displayed increased levels of phosphorylated 44-45 kDa Cx43, assessed by western blotting. In addition, FGF-2 significantly upregulated phosphorylation of the PKC target serines 262 and 368 on Cx43 at intercalated disks, assessed using phosphospecific antibodies in immunolocalization and western blotting assays. Our data show that FGF-2, administered by perfusion, can alter the phosphorylation status of Cx43 at cardiomyocyte intercalated disks, and suggest a link between phosphorylation of Cx43 at specific PKC sites and FGF-2 cardioprotection.

A cellular and molecular approach was used to gain new insight into the pathogenesis of interstitial fibrosis in chronic purine aminonucleoside nephrosis (PAN) nephrosis. Thirty experimental rats (PAN rats) were given 15 mg/100 g body wt of i.p. PAN at time 0, followed by 4.3 mg/100 g body wt i.p. on days <em>20</em>, 27 and 34; 25 control rats received i.p. saline at the same time intervals. All rats had a right unilateral nephrectomy within the first four days. Groups of control and PAN rats were killed at 21, 37, 52, 72 and 91 days. Renal sections were studied by immunofluorescence to quantitate interstitial macrophages, T lymphocytes and <em>fibroblasts</em>, and to characterize the deposition of the extracellular matrix (ECM) proteins (collagens I, III and IV, fibronectin and laminin) and the tissue inhibitor of the metalloproteinases (TIMP). Steady state concentrations of mRNA from the whole kidney for these ECM proteins, the metalloproteinases, TIMP, and transforming <em>growth</em> <em>factor</em> beta (TGF-beta 1) were quantitated by Northern blot analysis. Significant increases in the number of interstitial macrophages and T lymphocytes were found in the PAN rat groups compared to that in controls. All ECM proteins examined were quantitatively increased in the tubulo-interstitium of PAN rats. The pattern of distribution of some ECM proteins was also modified in experimental animals. TIMP was increased in the interstitium of PAN rats; at later times, TIMP was most prominent in sclerotic regions of the glomeruli and in tubular protein droplets. Northern blot analysis revealed increased steady-state mRNA levels for components of each of the ECM proteins, no change for the metalloproteinases--stromelysin or collagenase--and a marked increase for TIMP and TGF-beta 1 in PAN animals. The results of this study suggest that the diffuse interstitial fibrosis found in chronic PAN nephrosis results from both increased production of ECM proteins and decreased matrix degradation.

BACKGROUND

Discrepancy exists between the potent effects of therapeutic angiogenesis in laboratory animals and the marginal results observed in patients with advanced coronary artery disease. In vitro and small animal data suggest that angiogenesis may depend on locally available nitric oxide (NO), but the impact of endothelial dysfunction on therapeutic angiogenesis in the myocardium has been unclear. We compared the effects of clinically applicable angiogenesis methods in swine in which endothelial dysfunction was experimentally induced to that observed in normal swine.

RESULTS

Miniswine were fed either a regular (N=13) or hypercholesterolemic diet (N=13) for <em>20</em> weeks. Hypercholesterolemic swine showed coronary endothelial dysfunction on videomicroscopy. Animals from both groups received 100 microg of perivascular sustained-release <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 in the lateral myocardial territory, previously made ischemic by placement of an ameroid constrictor around the circumflex artery. After 4 weeks of FGF-2 therapy, lateral myocardial perfusion was significantly lower in hypercholesterolemic than in normocholesterolemic swine, both at rest and during pacing (0.44+/-0.04 versus 0.81+/-0.15 mL/min/g at rest, respectively; P=0.006; and 0.50+/-0.06 versus 0.71+/-0.10 mL/min/g during pacing; P=0.02). Hypercholesterolemic swine showed no net increase in perfusion from FGF-2 treatment. Endothelial cell density and FGF receptor-1 expression were significantly lower in the lateral territory of hypercholesterolemic versus normocholesterolemic animals.

CONCLUSIONS

The cardiac angiogenic response to FGF-2 treatment using clinically applicable methods was markedly inhibited in hypercholesterolemic swine with coronary endothelial dysfunction. These findings suggest that coronary endothelial dysfunction is major obstacle to the efficacy of clinical angiogenesis protocols and constitutes a target toward making angiogenesis more effective in patients with advanced coronary disease.

BACKGROUND

Activating FGFR2 mutations are found in 10-16% of primary endometrial cancers and provide an opportunity for targeted therapy. We assessed the safety and activity of dovitinib, a potent tyrosine-kinase inhibitor of fibroblast growth factor receptors, VEGF receptors, PDGFR-β, and c-KIT, as second-line therapy both in patients with FGFR2-mutated (FGFR2(mut)) endometrial cancer and in those with FGFR2-non-mutated (FGFR2(non-mut)) endometrial cancer.

METHODS

In this phase 2, non-randomised, two-group, two-stage study, we enrolled adult women who had progressive disease after first-line chemotherapy for advanced or metastatic endometrial cancer from 46 clinical sites in seven countries. We grouped women according to FGFR2 mutation status and gave all women dovitinib (500 mg per day, orally, on a 5-days-on and 2-days-off schedule) until disease progression, unacceptable toxicity, death, or study discontinuation for any other reason. The primary endpoint was proportion of patients in each group who were progression-free at 18 weeks. For each group, the second stage of the trial (enrolment of 20 additional patients) could proceed if at least eight of the first 20 treated patients were progression free at 18 weeks. Activity was assessed in all enrolled patients and safety was assessed in all patients who received at least one dose of dovitinib. The completed study is registered with ClinicalTrials.gov, number NCT01379534.

RESULTS

Of 248 patients with FGFR2 prescreening results, 27 (11%) had FGFR2(mut) endometrial cancer. Between Feb 17, 2012, and Dec 13, 2013, we enrolled 22 patients in the FGFR2(mut) group and 31 patients in the FGFR2(non-mut) group. Seven (31·8%, 95% CI 13·9-54·9) patients in the FGFR2(mut) group and nine (29·0%, 14·2-48·0) in the FGFR2(non-mut) group were progression-free at 18 weeks. On the basis of predefined criteria, neither group continued to stage two: seven (35%) of the first 20 patients in the FGFR2(mut) group were progression free at 18 weeks, as were five (25%) of the first 20 in the FGFR2(mut) population. Rates of treatment-emergent adverse events were similar between groups and events were most frequently gastrointestinal. Overall, the most common grade 3 or 4 adverse events suspected to be related to the study drug were hypertension (nine patients; 17%) and diarrhoea (five; 9%). The most frequently reported serious adverse events suspected to be related to study drug were pulmonary embolism (four patients; 8%), vomiting (four; 8%), dehydration (three; 6%), and diarrhoea (three; 6%). Only one death was deemed to be treatment-related: one patient in the FGFR2(non-mut) group died from cardiac arrest with contributing reason of pulmonary embolism (grade 4, suspected to be study drug related) 4 days previously.

CONCLUSIONS

Second-line dovitinib in FGFR2(mut) and FGFR2(non-mut) advanced or metastatic endometrial cancer had single-agent activity, although it did not reach the prespecified study criteria. Observed treatment effects seemed independent of FGFR2 mutation status. These data should be considered exploratory and additional studies are needed.

BACKGROUND

Novartis Pharmaceuticals.

Interstitial fibrosis is a common outcome of longterm ureteral obstruction. One pathological arm of the fibrotic reaction in diverse tissue loci and experimental models is the retraction of granulation tissue. The role of the myofibroblast in granulation tissue contraction and fibrocontractive diseases has been well established, but the mechanisms leading to differentiation of fibroblastic cells into myo<em>fibroblasts</em> during the evolution of inflammation are not yet fully clarified. Investigators using other model systems have shown that macrophage-derived transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) may be pivotal in the process of myofibroblast modulation. Our laboratory has shown that the unilateral ureteral obstruction in the rat is characterized by a <em>20</em>-fold increment in infiltrating renal cortical interstitial macrophages, an increase in cortical TGF-beta 1 gene expression, which parallels the infiltrating macrophage burden, and immunolocalization of this peptide <em>growth</em> <em>factor</em> in close proximity to resident interstitial <em>fibroblasts</em>. Because of this model's features, it was our aim to assess whether a myofibroblastic modulation was operant in the renal cortex of obstructed rat kidneys versus the control contralateral unobstructed kidney specimens. Immunolabeling for alpha-smooth muscle actin and the intermediate filament protein, desmin, was detected and steadily intensified from 24 to 96 hours after unilateral ureteral obstruction in obstructed kidneys only. In temporal concert with the detection of alpha-smooth muscle actin protein, the mRNA expression for this cytoskeletal component exhibited 3.7-, 15.7-, and 4.1-fold increments in the renal cortex of obstructed kidneys versus the contralateral unobstructed kidney specimens at 24, 48, and 96 hours after unilateral ureteral obstruction, respectively. Whole body X-irradiation, administered to rats 11 days before proximal left ureteral ligation, significantly lowered cortical interstitial macrophage number, cortical TGF-beta and alpha-smooth muscle actin mRNA levels as well as the intensity of immunolabeling for alpha-smooth muscle actin from 12 to 96 hours after unilateral ureteral obstruction. These data support a postulate that renal cortical TGF-beta 1, derived from the infiltrating macrophage, in part, contributes to the subsequent interstitial fibrosis response to renal injury by fostering the modulation of <em>fibroblasts</em> to myo<em>fibroblasts</em> within the renal cortex after ureteral obstruction.

We have asked how posterior neural tissue is patterned in Xenopus by assaying the involvement of endogenous retinoic acid (RA) in this process and by using the labial Hox gene, HoxD1, as a posterior marker. Although RA is able to inhibit anterior gene expression and activate expression of more posterior genes, the normal role of retinoids in anteroposterior (A/P) patterning is unclear. HoxD1 is an early posterior neurectodermal marker, expressed from midgastrula with a later anterior expression limit in the future hindbrain. We previously showed that HoxD1 was induced as an immediate early response to retinoic acid in naive ectoderm (animal caps). Here, we use a truncated RARalpha2.2 receptor (RARDelta) to dominantly interfere with retinoid signaling. In embryos injected with RARDelta expression of HoxD1 is eliminated. Conjugates of ectoderm and dorsolateral mesoderm show that retinoid receptors are required in the ectoderm for HoxD1 induction. Further, expression of Krox-<em>20</em> in r3 and r5 of the presumptive hindbrain is compressed into a single stripe that suggests elimination of r5. RARalpha2.2 expression almost precisely overlaps that of HoxD1, suggesting that this receptor may normally activate HoxD1. Expression of neither more anterior genes including cement gland, forebrain, and midbrain markers nor a more posterior spinal cord marker is affected by RARDelta. These data suggest that the posterior hindbrain is the region of the nervous system most sensitive to retinoid loss. Finally, we compare the ability of RA and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) to posteriorize isolated anterior neurectoderm and show that both <em>factors</em> can act directly on this substrate. RA acts in a more anterior domain than does FGF; however, neither <em>factor</em> is equivalent to the natural posteriorizing capacity of the posterior mesoderm. We propose that endogenous retinoid and FGF signals pattern largely nonoverlapping regions along the A/P axis and that posterior neural patterning requires multiple inducers.

Chronic venous disease with skin changes of the leg is a common condition affecting up to 1 in <em>20</em> people in westernized countries. The causes of this problem are not fully understood, although research in recent years has revealed a number of important mechanisms that contribute to the disease process. Patients with chronic venous disease suffer persistently raised pressures in their deep and superficial veins in the lower limb. Leucocytes become "trapped" in the circulation of the leg during periods of venous hyper-tension produced by sitting or standing. Studies of the plasma levels of neutrophil granule enzymes shows that these are increased during periods of venous hypertension, suggesting that this causes activation of the neutrophils. Investigation of the leucocyte surface ligands CD11b and CD62L shows that the more activated neutrophils and monocytes are sequestered during venous hypertension. Measurement of plasma levels of the soluble parts of the endothelial adhesion molecules VCAM, ICAM, and ELAM show that these are all elevated in patients with chronic venous disease compared to controls. Following 30 minutes of venous hypertension produced by standing, these levels are further increased. These data suggest that venous hypertension causes neutrophil and monocyte activation, which in turn causes injury to the endothelium. Chronic injury to the endothelium leads to a chronic inflammatory condition of the skin that we know clinically as lipodermatosclerosis. This is mediated by perivascular inflammatory cells, principally macrophages, in the skin microcirculation. These stimulate <em>fibroblasts</em> in the skin leading to tissue remodeling and laying down of fibrous tissue. Vascular endothelial <em>growth</em> <em>factor</em> stimulates proliferation of capillaries within the skin. Skin in this state has the potential to ulcerate in response to minor injury.

Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic <em>growth</em> <em>factors</em> involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72 h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (<em>20</em>% compression, 1 Hz) over 3 days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24 h to evaluate the persistence of the effects. <em>Growth</em> <em>factor</em> (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial <em>growth</em> <em>factor</em> receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and transforming <em>growth</em> <em>factor</em> beta1 (TGF-beta1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) or platelet-derived <em>growth</em> <em>factor</em> (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24 h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical stimulation; this was significant in haematomas from younger, but not from older patients. Other GFs were not mechanically regulated. In conclusion, the paracrine pro-angiogenic capacity of early human haematomas is enhanced by mechanical stimulation. This effect lasts even after removing the mechanical stimulus and appears to be VEGFR2-dependent.

Increasing evidence shows the existence of nonproliferation-specific gene(s) whose expression is mostly present in <em>growth</em>-arrested cells. One member of this gene family has been identified by previous work as a nuclear protein of 57,000 Da, termed statin. Logical extensions of statin research are to identify the genomic and cDNA clones encoding for statin and to study the regulation of statin gene expression. During the search for the statin gene, we have identified a cDNA clone and a genomic clone named S1 and S10, respectively, by screening a rat brain lambda gt11 expression library with the statin antibody and subsequently using S1 cDNA as a probe to screen a rat genomic cosmid library. Here, we report the cloning and sequencing of the S1 cDNA and S10 genomic clones. Primary sequence analyses indicate that the derived amino acid sequence of S1 shares high homology (greater than 92.6%) with human elongation <em>factor</em> 1 alpha (EF-1 alpha), whereas the 5'- and 3'-untranslated regions are less than <em>20</em>% homologous. Despite the unusually high degree of similarity between S1 and human EF-1 alpha at the amino acid sequence level, their protein products are different and immunologically distinct. The in vitro transcription and translation product of S1 (pS1), a 49,000-Da polypeptide, reacts only with the monoclonal antibody against statin; this antibody exhibits no antigenic reaction to the EF-1 alpha protein. Northern blot analysis shows that the S1 message is most abundant in G0 phase of 3T3 mouse <em>fibroblasts</em>, but becomes significantly reduced in G1 and S phase cells. EF-1 alpha messages do not show such dramatic changes during cell cycle phase transition. These findings suggest that the expression of the identified S1 cDNA clone is specific for nonproliferating cells and that the in vitro translation product of the S1 cDNA is recognized by the statin antibody. Genomic Southern blots indicate that S1 cDNA is encoded by a single copy gene in the rat genome and is a unique member of the EF-1 alpha/S1 supermultigene family. DNA sequence analysis demonstrates that the rat S1 transcription unit is 12 kilobase pairs in length and contains seven introns. The organization of exons is virtually identical between S1 and human EF-1 alpha. In contrast, neither a TATA box nor a CAAT box is located in the proximal 5'-flanking regions from positions -1 to -1359 of the S1 gene, where we could expect to find the regulatory region containing the elements controlling gene expression; no evident sequence homology to the human EF-1 alpha gene is detected in this region.(ABSTRACT TRUNCATED AT 400 WORDS)

The development of a hormone-responsive glucose transport activity during differentiation of 3T3-L1 murine <em>fibroblasts</em> to an insulin-sensitive adipocyte-like phenotype was studied. Glucose transport activity was insensitive to insulin or insulin-like <em>growth</em> <em>factor</em> I (IGF-I) before differentiation, and was increased by 8-10-fold after differentiation by both insulin and IGF-I via their own respective receptors. In contrast, in undifferentiated cells insulin and IGF-I stimulated a large increase of [3H]thymidine incorporation into DNA via IGF-I receptors, indicating that undifferentiated 3T3-L1 cells are equipped with fully functioning hormone (IGF-I) receptors. Thus the previously described increase in expression of insulin receptors during differentiation cannot solely account for the development of hormone-sensitive glucose transport in the 3T3-L1 cell. The total glucose transport activity reconstituted from membrane fractions was increased by about 3-fold during differentiation. In differentiated cells, more than 80% of the total reconstitutable glucose transport activity was detected in an intracellular compartment (<em>20</em>0,000 g microsomes) as compared with about <em>20</em>% in undifferentiated cells. Immunoblots with specific antiserum confirmed previous reports indicating that the adipose tissue/muscle glucose transporter (GT3) was exclusively present in the differentiated cells, whereas the erythrocyte/brain glucose transporter (GT1) was detected in both differentiated and undifferentiated cells. Upon differentiation, GT1 was redistributed from plasma membranes to the intracellular compartment. In addition, the newly formed GT3 was predominantly found (greater than 80% of total) in the microsomal fraction of differentiated cells. Both GT1 and GT3 appeared to be hormone-sensitive, since in differentiated cells insulin as well as IGF-I gave rise to their translocation from the intracellular compartment to the plasma membrane. These data suggest that, in addition to the specific expression of the GT3 transporter, the formation of a large pool of intracellular glucose transporters comprising both GT1 and GT3 contributes to the development of insulin sensitivity in the 3T3-L1 cell.