BACKGROUND

At the present time, the pathogenesis of ovarian cancer remains poorly understood, with invasive diagnosis and ineffective treatment for women with the disease. Despite scientific and medical advances in oncology, the overall 5-year survival rate of 30% for ovarian cancer patients has not changed in 20 years. An understanding of the angiogenic process as it occurs in ovarian cancer would not only increase our knowledge of the pathogenesis of this cancer but also might offer novel opportunities for therapeutic intervention.

OBJECTIVE

Our aim was to study the expression of messenger RNA (mRNA) coding for four putative angiogenic factors in normal ovaries and benign and malignant ovarian tumors: platelet-derived endothelial cell growth factor (thymidine phosphorylase), vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1.

METHODS

Four normal ovaries and 25 tumors (seven benign, one of borderline malignancy, and 17 malignant) were collected from 29 patients during elective oophorectomy. The site of sampling (areas of high-velocity blood flow) was directed by transvaginal color Doppler imaging performed within 24 hours of the surgery. Increased blood flow within the tissues was demonstrated by the presence of color (i.e., the velocity was>> 7 cm/s) and, together with a pulsatile index of less than 1.0, constituted a positive scanning result. In scan-positive tissues, the area of maximum blood flow was chosen. In scan-negative tissues, a solid area was chosen in complex lesions, or the cyst wall was chosen in simple lesions. Ovarian RNA was subsequently extracted from areas of high-velocity flow (i.e., tissues with a positive scanning result) or from solid areas or septa in tissues with a negative scanning result. A ribonuclease protection assay was used to assess the expression of mRNA coding for the four angiogenic factors.

RESULTS

Two normal ovaries (containing a corpus luteum) and one benign and 17 malignant tumors (plus the borderline) gave a positive scanning result. There was a significant difference between the expression of mRNA for platelet-derived endothelial cell growth factor between scan-positive and scan-negative tissues (P < .001) and between benign and malignant tumors (P < .001).

CONCLUSIONS

Areas of high blood velocity in ovarian tumors are associated with increased expression of platelet-derived endothelial cell growth factor.

CONCLUSIONS

Drugs that affect the angiogenic activity of platelet-derived endothelial cell growth factor offer a potential route for therapeutic intervention.

We have identified and cloned a novel connective tissue <em>growth</em> <em>factor</em>-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity ( approximately 60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like <em>growth</em> <em>factor</em> binding domain, a von Willebrand <em>Factor</em> type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA ( approximately 1.3 kilobases) is expressed in primary human osteoblasts, <em>fibroblasts</em>, ovary, testes, and heart, and a approximately 26-kDa protein is secreted from primary human osteoblasts and <em>fibroblasts</em>. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of 125I-labeled insulin-like <em>growth</em> <em>factors</em> to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros <em>17</em>/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.

Pericardial fluid (PF) may contain myocardial <em>growth</em> <em>factors</em> that exert paracrine actions on cardiac myocytes. The aims of this study were (1) to investigate the effects of human PF and serum, collected from patients undergoing cardiac surgery, on the <em>growth</em> of cultured adult rat cardiac myocytes and (2) to relate the <em>growth</em> activity of both fluids to the adaptive changes in overloaded human hearts. Both PF and serum increased the rate of protein synthesis, measured by [14C]phenylalanine incorporation in adult rat cardiomyocytes (PF, +71.9 +/- 8.2% [n = <em>17</em>]; serum, +14.9 +/- 6.5% [n = 13]; both P < .01 versus control medium). The effects of both PF and serum on cardiomyocyte <em>growth</em> correlated positively with the respective left ventricular (LV) mass. However, the magnitude of change with PF was 3-fold greater than with serum (P < .01). These trophic effects of PF were mimicked by exogenous basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) and inhibited by anti-FGF2 antibodies and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), suggesting a relationship to FGF2. In addition, FGF2 concentration in PF was 20 times greater than in serum. On the other hand, the LV mass-dependent trophic effect, present in both fluids, was independent of FGF2 concentration or other <em>factors</em>, such as angiotensin II, atrial natriuretic <em>factor</em>, and TGF-beta. These data suggest that FGF2 in human PF is a major determining <em>factor</em> in normal myocyte <em>growth</em>, whereas unidentified LV mass-dependent <em>factor</em>(s), present in both PF and serum, participates in the development of ventricular hypertrophy.

Rearrangements of the genes encoding the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) and platelet-derived <em>growth</em> <em>factor</em> receptors (PDGFR) alpha or beta receptor tyrosine kinases are found in a rare but important subset of patients with atypical myeloproliferative disorders that are usually but not always associated with eosinophilia. Chromosomal translocations or other rearrangements at 8p11-12, 4q12 or 5q31-33 give rise to diverse fusion genes encoding chimaeric proteins with constitutive transforming activity. There is considerable molecular heterogeneity with 8 partner genes currently known for FGFR1, 6 for PDGFRA and <em>17</em> for PDGFRB. The vast majority of patients with PDGFRA or PDGFRB fusions achieve rapid and durable complete haematological and molecular responses to sustained imatinib therapy. A key ongoing challenge is to define the molecular pathogenesis of the great majority of atypical myeloproliferative disorders for whom the causative lesion remains unknown, since very few of these cases gain any benefit from imatinib or other second-generation inhibitors.

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived <em>growth</em> <em>factor</em> (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine <em>17</em> in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine <em>17</em> forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position <em>17</em> and examined the ability of these proteins to transform C127 <em>fibroblasts</em>, which express endogenous PDGF beta receptor. Although several position <em>17</em> mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position <em>17</em> of the E5 protein and threonine 513 of the receptor. The nature of the residue at position <em>17</em> also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position <em>17</em> of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position <em>17</em> in E5 dimerization and in complex formation between the E5 protein and the PDGF beta receptor.

Uterine leiomyomas are the most common tumors of the reproductive tract, afflicting women between the ages of 30--55 yr. Although considered to be the leading cause of hysterectomies in the United States, little is known of the etiology and mechanisms of pathogenesis in leiomyomas. Accordingly, rapid analysis of differential expression (RADE) was employed to identify genes that are abnormally expressed in leiomyomas. Of the several genes identified, Cyr61, a member of the CCN family of <em>growth</em> and angiogenic regulators, was shown to be markedly down-regulated at the messenger ribonucleic acid (mRNA) and protein levels in leiomyoma tumors compared with the matched uterine myometrial controls (n = 38). In addition, in situ hybridization experiments corroborated the lack of Cyr61 expression in leiomyoma cells, whereas abundant transcript levels were identified in adjacent myometrial smooth muscle cells. To elucidate the mechanisms of Cyr61 gene regulation in leiomyomas, we determined the effects of ovarian steroids, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and serum, on Cyr61 expression using an ex vivo culture system. Treatment of human myometrial explants with <em>17</em> beta-estradiol and bFGF up-regulated Cyr61 transcripts, whereas the progesterone receptor agonist, R5020 (alone or in combination with <em>17</em> beta-estradiol), had no effect. Paradoxically, neither <em>17</em> beta-estradiol nor bFGF was capable of up-regulating Cyr61 mRNA in leiomyoma explants despite elevated levels of ER alpha mRNA, suggesting a possible defect in steroid and <em>growth</em> <em>factor</em> regulation. Thus, dysregulation of Cyr61 by estrogen and bFGF may contribute to down-regulation of Cyr61 in leiomyomas, which, in turn, may predispose uterine smooth muscle cells toward sustained <em>growth</em>.

Pyramidal neurons in hippocampal subregions are selectively vulnerable in certain disease states. To investigate, we tested the hypothesis that selective vulnerability in human hippocampus is related to regional differences in neuronal cell death and cell receptor gene expression in CA1 vs. CA3 subregions. We used laser capture microdissection to remove approximately 600 CA1 and 600 CA3 pyramidal neurons each from five fresh-frozen normal post-mortem brains, extracted total RNA and double-amplified mRNA. This was reverse transcribed and labeled for hybridization onto human cDNA array chips containing probes to 10,<em>17</em>4 genes and unknown ESTs. RNA from additional microdissections was pooled for replicate hybridizations and quantitative RT-PCR validation. Gene expression differences were few (< 1%). We found 43 enriched genes in CA1 neuronal samples that included peripheral benzodiazipine receptor-associated protein, nicotinic cholinergic receptor, two chemokine receptors (CCR1 and CCR5) and several transcriptional <em>factors</em>. We found <em>17</em> enriched genes in the CA3 neuronal samples that included <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor and prostaglandin-endoperoxide synthase 1. We found no differential gene expression for 23 calcium channel proteins; nine transporter proteins; 55 cell death and apoptotic regulator proteins; and an additional 497 cell receptors, including 24 glutamate receptors. Quantitative RT-PCR of four differentially expressed genes confirmed the microarray data. The results confirm the ability to examine gene expression profiles in microdissected neurons from human autopsy brain. They show only minor gene expression differences between two distinct neuronal populations in the hippocampus and suggest that selective hippocampal vulnerability is due to <em>factors</em> other than intrinsic differential expression in glutamate receptors and cell death genes.

Nerve <em>growth</em> <em>factor</em> receptor (NGFR) is a transmembrane glycoprotein without intrinsic tyrosine kinase activity, whose expression is not restricted to neural cells. NGFR is reported to act as a tumour suppressor, negatively regulating cell <em>growth</em> and proliferation. NGFR expression was immunohistochemically analysed in normal breast tissue and in 140 benign, biphasic and preinvasive breast lesions, in 22 tumours with myoepithelial differentiation and in two cohorts of breast cancer patients: a series of 245 invasive breast carcinomas studied with tissue microarrays and 37 high-grade invasive ductal carcinomas with basal-like immunophenotype. NGFR consistently displayed membrane reactivity in myoepithelial cells arranged as a continuous layer around normal ducts and lobular units, intralobular <em>fibroblasts</em>, vascular adventitia and nerve bundles. Myoepithelial cells of benign proliferations and pre-invasive lesions were consistently positive for NGFR. Scattered NGFR-positive cells were observed in solid areas of six out of nine cases of hyperplasia of usual type, whereas in flat atypia, lobular carcinoma in situ and virtually all cases of ductal carcinoma in situ (97.5%), NGFR was restricted to the myoepithelial layer. Positivity for NGFR was observed in 11 out of 245 (4.5%) breast carcinomas, nine out of 20 (45%) metaplastic breast carcinomas and 14 out of 37 (38%) basal-like breast carcinomas. NGFR expression in invasive tumours significantly correlated with that of cytokeratins 5/6 (P<0.05), 14 (P<0.0001) and <em>17</em> (P<0.0005) and EGFR (P<0.0001) and displayed an inverse correlation with oestrogen and progesterone receptors (both, P<0.0001). NGFR showed a statistically significant association with longer disease-free (P<0.05) and overall survival (P<0.01) in the cohort of patients with basal-like carcinomas. This study demonstrates the usefulness of NGFR as a new adjunct marker to identify myoepithelial cells in preinvasive lesions and myoepithelial differentiation in breast carcinomas. Furthermore, provisional data in a small number of basal-like breast carcinomas suggest that NGFR may identify a subgroup of basal-like breast carcinomas with good prognosis.

Estrogen is known to modulate angiogenesis, both under physiological and pathological conditions, and has been demonstrated to augment angiogenesis induced by bFGF in a mouse model. We have modified this mouse model and measured the apparent plasma volume in Matrigel plugs containing basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in wild type and estrogen receptor knockout, ovariectomized mice in the presence and absence of exogenous <em>17</em> beta estradiol. The apparent plasma volume was determined by measuring the fluorescence of the excised plug 10 min. after injection of fluoroscein labeled dextran 150. In wild type mice exogenous <em>17</em> beta estradiol increased the apparent plasma volume of the Matrigel plug and the uterine weight significantly. In the estrogen receptor knockout mice exogenous <em>17</em> beta estradiol caused a small, but significant increase in uterine weight but was without effect on the apparent plasma volume of the Matrigel plug. It is concluded that functional estrogen receptors are essential for the augmentation of bFGF-induced angiogenesis by exogenous <em>17</em> beta estradiol in female mice.

At Day <em>17</em> of in ovo development, chondrocyte hypertrophy including synthesis of collagen X takes place in a limited region within the cranial part of chick embryo sternum. Here we analyze in suspension culture the differences in response to single <em>growth</em> <em>factors</em> of chondrocytes derived from the cranial part versus cells derived from the caudal part. Cells from either part were cultured separately without serum in the presence of insulin-like <em>growth</em> <em>factor</em>-1, transforming <em>growth</em> <em>factor</em> beta 2, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, or thyroid hormones. In culture, chondrocytes derived from the cranial part of sterna from 14- to 18-day-old chicken embryos become hypertrophic and initiated the synthesis of collagen X and alkaline phosphatase. These processes were enhanced by anabolic diffusible signals, such as those contained in fetal bovine serum, insulin-like <em>growth</em> <em>factor</em>-1, or thyroxine. Cells derived from the caudal part lack this capacity and, instead, prevented hypertrophy of cranial cells in cocultures, presumably by secreting diffusible signals. As candidate molecules, we have identified transforming <em>growth</em> <em>factor</em> beta 2 and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, which both were released by chondrocytes. Synergistic action of transforming <em>growth</em> <em>factor</em> beta 2 and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was required to suppress insulin-like <em>growth</em> <em>factor</em>-1-stimulated maturation of cranial chondrocytes in culture.

BACKGROUND

Arresting or regressing kidney scarring is of major clinical relevance. Platelet-derived growth factor D (PDGF-D) is widely expressed in fibrotic kidneys. Administration of the PDGF-D neutralizing fully human monoclonal antibody CR002 in the acute phase of progressive anti-Thy 1.1 glomerulonephritis reduced glomerular and secondary tubulointerstitial damage.

METHODS

Using this model, we now assessed the effects of CR002 (n=15) vs irrelevant control IgG (n=17) administered on days 17, 28 and 35 after disease induction, i.e. after acute glomerular damage had subsided.

RESULTS

In vitro, CR002 inhibited the PDGF-D- but not the PDGF-B-induced proliferation of rat renal fibroblasts. Following the first CR002 injection on day 17, exposure to therapeutic levels was maintained until day 49. Proteinuria in the CR002-treated group was transiently reduced between days 49 and 77 (-19 to -23% in comparison with the controls; P<0.05). On day 100, CR002 treatment reduced the number of rats that had doubled their serum creatinine (CR002: 40 vs controls: 71%; P<0.05). Compared with controls, the CR002 animals, on day 100, significantly lowered glomerular expression of vimentin and collagens as well as tubulointerstitial damage scores, interstitial fibrosis, vimentin and cortical PDGF-D mRNA levels.

CONCLUSIONS

PDGF-D antagonism, even after the phase of acute glomerular damage, exerts beneficial effects on the course of tubulointerstitial damage, i.e. the final common pathway of most renal diseases.

<em>Growth</em> hormone receptor (GHR) is a cytokine receptor superfamily member that binds <em>growth</em> hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis <em>factor</em>-alpha-converting enzyme (ADAM-<em>17</em>) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived <em>growth</em> <em>factor</em> has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected <em>fibroblasts</em> expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic <em>fibroblasts</em> derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.

OBJECTIVE

Studies in avian models of myopia have shown that refractive error development can be influenced by exogenously delivered fibroblast growth factor (FGF)-2. The present study sought to determine whether endogenous FGF-2 was associated with retinoscleral signalling or scleral remodelling during changes in refractive error in a mammalian model of myopia.

METHODS

Myopia was induced in tree shrews over a 5-day period. One group of animals was then allowed 3 days of recovery from the induced myopia. Endogenous levels of FGF-2 were measured in scleral and retinal homogenates using ELISA. Real-time PCR was used to investigate scleral FGF-2 and FGF receptor (FGFR)-1 mRNA expression.

RESULTS

No difference in FGF-2 content was found in posterior scleral or retinal extracts of myopic eyes (scleral -4+/-9%, retinal +23+/-17%) or recovering eyes (scleral -10+/-18%, retinal +1+/-13%), when compared with contralateral control eyes. In addition, no significant changes were found in scleral FGF-2 mRNA expression in myopic or recovering eyes (+106+/-56% and +14+/-12% respectively, P=0.21). However, FGF-2 concentration was significantly higher in anterior, relative to posterior, scleral regions in all animals (1602+/-105 vs 1030+/-50pg/mg respectively P<0.001). Expression of scleral FGFR-1 mRNA was upregulated in myopic eyes (+186+/-32%, P=0.01) but returned to control eye levels during recovery (+63+/-20%).

CONCLUSIONS

The findings indicate that alterations in endogenous retinal or scleral FGF-2 levels are not associated with changes in scleral remodelling in this mammalian model of myopia. However, the reversible changes found in FGFR-1 expression in the sclera of myopic eyes mean that an indirect role for FGF-2 in the control of scleral remodelling is implicated. The anteroposterior difference found in scleral FGF-2 concentration indicates a role for this cytokine in the control of normal scleral growth and development and, presumably, eye size.

OBJECTIVE

Numerous studies have evaluated the prevalence and importance of vitamin D deficiency among patients with chronic kidney disease and end-stage renal disease; however, little is known about vitamin D levels in acute kidney injury (AKI). We evaluated the association between vitamin D metabolites and clinical outcomes among patients with AKI.

METHODS

Prospective cohort study.

METHODS

A total of 30 participants with AKI and 30 controls from general hospital wards and intensive care units at a tertiary care hospital were recruited for the study.

METHODS

Plasma levels of 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2 D], 24R,25-dihydroxyvitamin D3 , vitamin D binding protein (VDBP) and fibroblast growth factor 23 (FGF23) were measured within 24 hours of AKI onset and 5 days later. Bioavailable 25(OH)D and 1,25(OH)2 D levels, defined as the sum of free- and albumin-bound 25(OH)D and 1,25(OH)2 D, were estimated using equations.

RESULTS

Compared to controls, participants with AKI had lower levels of 1,25(OH)2 D [17 (10-22) vs 25 (15-35) pg/ml, P = 0·01], lower levels of VDBP [23 (15-31) vs 29 (25-36) mg/dl, P = 0·003] and similar levels of bioavailable 25(OH)D and 1,25(OH)2 D at enrolment. Levels of bioavailable 25(OH)D were inversely associated with severity of sepsis in the overall sample (P < 0·001). Among participants with AKI, bioavailable 25(OH)D, but not other vitamin D metabolites, was significantly associated with mortality after adjusting for age and serum creatinine (adjusted odds ratio per 1 SD ln [bioavailable 25(OH)D] = 0·16, 95% confidence interval = 0·03-0·85).

CONCLUSIONS

Bioavailable 25(OH)D could have a role as a biomarker or mediator of adverse outcomes among patients with established AKI.

<AbstractText>X-linked hypophosphataemia in children is characterised by elevated serum concentrations of <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), hypophosphataemia, rickets, lower extremity bowing, and <em>growth</em> impairment. We compared the efficacy and safety of continuing conventional therapy, consisting of oral phosphate and active vitamin D, versus switching to burosumab, a fully human monoclonal antibody against FGF23, in paediatric X-linked hypophosphataemia.</AbstractText><AbstractText>In this randomised, active-controlled, open-label, phase 3 trial at 16 clinical sites, we enrolled children with X-linked hypophosphataemia aged 1-12 years. Key eligibility criteria were a total Thacher rickets severity score of at least 2·0, fasting serum phosphorus lower than 0·97 mmol/L (3·0 mg/dL), confirmed PHEX (phosphate-regulating endopeptidase homolog, X-linked) mutation or variant of unknown significance in the patient or a family member with appropriate X-linked dominant inheritance, and receipt of conventional therapy for at least 6 consecutive months for children younger than 3 years or at least 12 consecutive months for children older than 3 years. Eligible patients were randomly assigned (1:1) to receive either subcutaneous burosumab starting at 0·8 mg/kg every 2 weeks (burosumab group) or conventional therapy prescribed by investigators (conventional therapy group). Both interventions lasted 64 weeks. The primary endpoint was change in rickets severity at week 40, assessed by the Radiographic Global Impression of Change global score. All patients who received at least one dose of treatment were included in the primary and safety analyses. The trial is registered with ClinicalTrials.gov, number NCT02915705.</AbstractText><AbstractText>Recruitment took place between Aug 3, 2016, and May 8, 20<em>17</em>. Of 122 patients assessed, 61 were enrolled. Of these, 32 (18 girls, 14 boys) were randomly assigned to continue receiving conventional therapy and 29 (16 girls, 13 boys) to receive burosumab. For the primary endpoint at week 40, patients in the burosumab group had significantly greater improvement in Radiographic Global Impression of Change global score than did patients in the conventional therapy group (least squares mean +1·9 [SE 0·1] with burosumab vs +0·8 [0·1] with conventional therapy; difference 1·1, 95% CI 0·8-1·5; p<0·0001). Treatment-emergent adverse events considered possibly, probably, or definitely related to treatment by the investigator occurred more frequently with burosumab (<em>17</em> [59%] of 29 patients in the burosumab group vs seven [22%] of 32 patients in the conventional therapy group). Three serious adverse events occurred in each group, all considered unrelated to treatment and resolved.</AbstractText><AbstractText>Significantly greater clinical improvements were shown in rickets severity, <em>growth</em>, and biochemistries among children with X-linked hypophosphataemia treated with burosumab compared with those continuing conventional therapy.</AbstractText><AbstractText>Ultragenyx Pharmaceutical and Kyowa Kirin International.</AbstractText>

Angiogenesis is critical in melanoma progression and metastasis and relies on the synthesis and release of proangiogenic molecules such as vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-A and <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs). S100A13 is a small calcium-binding protein that facilitates the release of FGF-1, the prototype of the FGF family. S100A13 is upregulated in astrocytic gliomas, in which it correlates with VEGF-A expression, microvessel density and tumor grading, and promotes a more aggressive, invasive phenotype in lung cancer-derived cell lines. To investigate the involvement of S100A13 in human cutaneous melanoma, we analyzed a series of 87 cutaneous melanocytic lesions: 14 common acquired melanocytic nevi, 14 atypical, so-called 'dysplastic' nevi, 45 melanomas (<em>17</em> radial <em>growth</em> phase and 28 vertical <em>growth</em> phase) and 14 melanoma metastases. Main clinical and pathological features, including histotype, Breslow thickness, Clark's level and outcome were recorded. Microvessel density was determined with CD105/endoglin staining. Semiquantitative determination of S100A13, FGF-1 and VEGF-A protein expression was obtained by immunostaining. Quantification of S100A13 mRNA was achieved by real-time PCR. We found that S100A13 was expressed in melanocytic lesions; compared with benign nevi, S100A13 protein expression was significantly upregulated in melanomas (P=0.024), in which it correlated positively with the intensity of VEGF-A staining (P=0.041) and microvessel density (P=0.007). The level of expression of S100A13 mRNA also significantly increased with progression of disease, from radial <em>growth</em> phase (0.7+/-0.7) to vertical <em>growth</em> phase (3.6+/-3.1) to metastases (7.0+/-7.0) (P<0.001). Furthermore, S100A13 mRNA correlated positively with VEGF-A (P=0.023), TNM stage (P=0.05), risk of relapse (P=0.014) and status at follow-up (P=0.024). In conclusion, S100A13 is expressed in melanocytic lesions when the angiogenic switch occurs and it may cooperate with VEGF-A in supporting the formation of new blood vessels, favoring the shift from radial to vertical tumor <em>growth</em>. Therefore, S100A13 may represent a new angiogenic and prognostic marker in melanoma.

OBJECTIVE

There have been several case reports of improvement in the appearance of mature burn scars following treatment with fractional CO(2) lasers. However, the biochemical mechanisms responsible for these improvements have not been elucidated.

METHODS

Ten patients with mature, full-thickness, hypertrophic burn scars received initial treatment with a fractional CO(2) laser. Clinical improvement was measured with Vancouver Scar Scale as well as Patient and Observer Scar Assessment Scale. Fresh tissue samples were obtained before the initial treatment and 48 hours after the first treatment for TaqMan Real-time RT-PCR analyses. Expressions of several scar-related biological markers, including types I and III procollagen, matrix metalloproteinase (MMP)-1, -13, transforming <em>growth</em> <em>factor</em> (TGF)-β1, β2, β3, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), as well as microRNA miR-<em>17</em>-92 cluster, were investigated.

RESULTS

There were significant improvements in both observer and subject ratings in all scales. Both types I and III procollagen mRNA levels were dramatically down-regulated after treatment, but the ratio of types I/III procollagen mRNA was not different. The expression of MMP-1 was significantly up-regulated after treatment, while TGF-β2, -β3, and bFGF levels were significantly down-regulated. Expression of miR-18a and miR-19a were dramatically up-regulated (P < 0.05) after treatment.

CONCLUSIONS

Our study indicated that fractional CO(2) resulted in clinical improvement of mature burn scar. Alteration of types I and III procollagen, MMP-1, TGF-β2, -β3, bFGF, as well as miRNAs miR-18a and miR-19a expression may be responsible for the clinical improvement after treatment. Our finding may have implications for novel treatments and further our understanding of fractional CO(2) laser treatment.

Drugs that inhibit <em>growth</em> of tumours and their blood supply could have considerable therapeutic potential. 2-Methoxyoestradiol-3,<em>17</em>-O,O-bis-sulphamate (2-MeOE2bisMATE) has been shown to inhibit the proliferation of MCF-7 (ER+) breast cancer cells and angiogenesis in vitro. 2-MeOE2bisMATE and its analogue, <em>17</em>-Cym-2-MeOE2MATE, were investigated for their ability to inhibit in vivo angiogenesis and tumour <em>growth</em>. The mouse Matrigel plug assay for angiogenesis was used to investigate the effect of compounds on neovascularisation and was quantified using a FITC-dextran injection technique. Nude mice bearing tumours derived from MCF-7 cells were used to assess efficacy on tumour <em>growth</em>. Tumour sections were stained for VEGFR-2 and Ki67 to assess tumour angiogenesis and cell proliferation respectively. Matrigel plugs supplemented with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> resulted in increased neovascularisation over 7 days. Oral administration of 2-MeOE2bisMATE for 7 days at 10 or 50 mg kg(-1) significantly reduced neovascularisation to or below control levels respectively. <em>17</em>-Cym-2-MeOE2MATE at 20 mg kg(-1) was equally effective. 2-MeOE2bisMATE, dosed daily for 21 days, caused a 52% reduction in tumour <em>growth</em> at 5 mg kg(-1) and 38% regression at 20 mg kg(-1). <em>17</em>-Cym-2-MeOE2MATE (20 mg kg(-1)) reduced tumour <em>growth</em> by 92%. Immunohistochemistry revealed a reduction in angiogenesis and proliferation. Matrigel plug and tumour imaging after FITC-dextran injection indicated that 2-MeOE2bisMATE caused a marked disruption of vasculature. These sulphamoylated oestrogen derivatives have been shown to be potent inhibitors of angiogenesis in vivo. This, together with their ability to inhibit tumour <em>growth</em>, indicates the potential of this new class of drugs for further development for cancer therapy.

BACKGROUND

Atrioventricular (AV) nodal ablation for management of atrial fibrillation (AF) is irreversible and requires permanent pacemaker implantation. We hypothesized that as an alternative, implantation of autologous fibroblasts in the perinodal region would focally modify AV nodal conduction and that this modulation would be enhanced by pretreatment with transforming growth factor-beta1 (TGF-beta1), a stimulant of fibroblasts.

RESULTS

Skin biopsies were taken from 12 mongrel dogs, and derived fibroblasts were dissociated and grown in culture for 2 weeks. Multiple injections (0.25 mL) were made through an 8F NOGA catheter along the fast/slow AV nodal pathways as guided by an electroanatomic mapping system. Seven dogs received fibroblasts alone (1x10(6) cells/mL), 7 dogs received TGF-beta1 (5 microg), 4 dogs received fibroblasts and TGF-beta1 (1x10(6) cells/mL+5 microg), and 4 dogs received saline only. AV node function was assessed at baseline and after 4 weeks. Saline (80 mL) with assigned therapy (0.25 mL per injection) was injected into the peri-AV nodal region in each dog. At baseline, the AH interval (66+/-3 ms) and the average RR interval (331+/-17 ms) in pacing-induced AF were similar in each cohort. The increase in AH interval in normal sinus rhythm was longer after fibroblast (23+/-4 versus 5+/-5 ms; P=0.05) and fibroblast plus TGF-beta1 (50+/-5 versus 5+/-5 ms; P<0.001) injections than with saline alone, with similar findings during high right atrium and distal coronary sinus pacing. The AH interval was not significantly increased after TGF-beta1 injections. The AH interval was significantly longer after fibroblast plus TGF-beta1 injections than with either therapy (TGF-beta1 or fibroblasts) alone. The RR interval during AF was increased in dogs that received fibroblasts alone (110+/-36 versus -41+/-34 ms) and to a greater extent with the addition of TGF-beta1 (294+/-108 versus -41+/-34 ms). No AV block was seen in any cohort at 4 weeks. Labeled fibroblasts that expressed vimentin were identified in all dogs that received cell injections at 4 weeks.

CONCLUSIONS

AV nodal modification can be achieved with injected fibroblasts without the creation of AV block. The effect on AV node conduction is substantially enhanced by pretreatment of fibroblasts with TGF-beta1. These data have therapeutic potential for the management of rapid ventricular rate during AF without pacemaker implantation.

The aim of this study was to identify the cardiac oxidoreductases involved in the metabolism of 4-hydroxy-2-trans-nonenal (HNE), an alpha,beta unsaturated aldehyde generated during the peroxidation of omega-6 polyunsaturated fatty acids. In homogenates of bovine, human and rat ventricles the primary pyridine coenzyme-linked metabolism of HNE was associated with NADPH oxidation. The NADPH-dependent enzyme catalysing HNE reduction was purified to homogeneity from bovine heart. The purified enzyme displayed kinetic and immunological properties identical with the polyol pathway enzyme aldose reductase (AR), and catalysed the reduction of HNE to its alcohol 1,4-dihydroxynonene (DHN), with a Km of 7+/-2 microM. In the presence of NADP the enzyme did not catalyse the oxidation of DHN. During catalysis, HNE did not cause inactivation of AR. Nevertheless when the apoenzyme was incubated with HNE a dissociable complex was formed between the enzyme and HNE, followed by irreversible loss of activity. Inactivation of the enzyme by HNE was prevented by NADP. Partial modification of the enzyme with HNE led to a <em>17</em>-fold increase in the KHNEm and Kglyceraldehydem, and the HNE-modified enzyme had a 500-fold higher IC50 for sorbinil than for the reduced enzyme, whereas the IC50 for tolrestat increased 25-fold. Incubation of the enzyme with radiolabelled HNE resulted in the incorporation of 2 mol of the aldehyde per mol of the enzyme. Sequence analysis of the radiolabelled peptides revealed modification of Cys-298 and Cys-187. The amino acid sequence of the HNE-modified peptides confirmed that the HNE-reducing cardiac enzyme is AR and not a related protein such as the <em>fibroblast</em>-<em>growth</em>-<em>factor</em>-regulated protein FR-1 or the mouse vas deferens protein MVDP. These results indicate that AR represents the only major oxidoreductase in the heart capable of utilizing HNE. The high affinity of the enzyme for HNE, the lack of inactivation during catalysis, and the lack of significant alcohol dehydrogenase activity of the protein suggests that AR-mediated catalysis of HNE is unlikely to be limited by substrate/product inhibition. Thus AR might constitute an antioxidative enzyme involved in myocardial protection against endogenous and exogenous cytotoxic aldehydes and against oxidative stress.

The Caenorhabditis elegans EGL-<em>17</em>/FGF protein is involved in the gonadal signaling that guides the migrations of sex myoblasts (SMs). egl-<em>17</em>::GFP reporter constructs are expressed dynamically in vulval cell lineages. Expression in the primary vulval cells is correlated with the precise positioning of SMs. We have investigated the cis-regulatory requirements for cell- and stage-specific expression of egl-<em>17</em>. Three enhancer elements that specify the expression of the egl-<em>17</em>::GFP reporter gene in primary or secondary vulval cells at certain stages were identified. Sequence analysis has suggested a number of potential transcription <em>factor</em> binding sites within the enhancer elements. egl-<em>17</em> is most likely a direct target of the LIN-39 Hox protein because mutations either in the lin-39/hox gene or at the consensus HOX/PBC binding site within the distal enhancer of the egl-<em>17</em> gene eliminated distal enhancer-activated egl-<em>17</em> expression. Since expression of egl-<em>17</em>::GFP driven by the distal enhancer can no longer be turned off at late stages in lin-1 and lin-31 mutants, egl-<em>17</em> may also be regulated by Ras signaling through repression of LIN-1 and LIN-31 activities. Interspecies transformation experiments showed that egl-<em>17</em> cis-regulatory elements are structurally and functionally conserved between C. elegans and C. briggsae.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) regulate bone <em>growth</em>, but their expression in human cartilage is unclear. Here, we determined the expression of entire FGF family in human fetal <em>growth</em> plate cartilage. Using reverse transcriptase PCR, the transcripts for FGF1, 2, 5, 8-14, 16-19, and 21 were found. However, only FGF1, 2, <em>17</em>, and 19 were detectable at the protein level. By immunohistochemistry, FGF<em>17</em> and 19 were uniformly expressed within the <em>growth</em> plate. In contrast, FGF1 was found only in proliferating and hypertrophic chondrocytes whereas FGF2 localized predominantly to the resting and proliferating cartilage. In addition, only the 18 kD isoform of FGF2 was found in resting chondrocytes while proliferating chondrocytes also synthesized 22 kD and 24 kD FGF2, similar to in vitro cultivated chondrocytes. In cell <em>growth</em> experiments, FGF1, 2, and <em>17</em> but not FGF19 inhibited the proliferation of FGFR3-expressing rat chondrosarcoma chondrocytes (RCS) with relative potency FGF2>>) FGF1 = FGF<em>17</em>. We conclude that FGF1, 2, <em>17</em>, and 19 are the predominant FGF ligands present in developing human cartilage that are, with the exception of FGF19, experimentally capable of inhibiting chondrocyte proliferation.

The process of decidualization involves the morphological and functional transformation of stromal <em>fibroblasts</em> to decidual cells. The objective of this study was to define appropriate in vitro culture conditions required for decidualization of baboon stromal cells. Parallel studies were also done with human endometrial stromal cells for comparative analysis. Human stromal cells produced prolactin and insulin-like <em>growth</em> <em>factor</em>-binding protein (IGFBP)-1 in response to hormones (estradiol-<em>17</em>beta [36 nM], medroxyprogesterone acetate [1 microM], and relaxin [100 ng/ml]), and production was enhanced in the presence of 0.1 mM dibutyryl cAMP (dbcAMP). By contrast, baboon cells did not produce any detectable levels of prolactin, even in the presence of hormones and dbcAMP. IGFBP-1 expression in baboon stromal cells was detectable by Day 6 of hormone and dbcAMP treatment and increased exponentially thereafter. In both human and baboon stromal cells, alpha smooth muscle actin (alphaSMA) expression, an early marker for decidualization in the baboon in vivo, was induced spontaneously under normal culture conditions. Furthermore, a decrease in alphaSMA expression was observed in cells producing high levels of IGFBP-1. Human cells produced significant levels of IGFBP-1 (p < or = 0.01) in response to short-term dbcAMP treatment (48 h) after 2 and 12 days of hormone treatment. However, baboon stromal cells required <em>17</em> days of hormonal treatment before cells became responsive to short-term dbcAMP treatment (p < or = 0.01). Finally, human endometrial stromal cells expressed the protein kinase A regulatory subunits RIalpha, RIbeta, RIIalpha, and RIIbeta whereas baboon stromal cells expressed RIalpha, RIIalpha, and RIIbeta. No difference in the mRNA expression of these isoforms was observed in decidualized or nondecidualized cells of either human or baboon endometrium. Our observations indicate that baboon stromal cells can be induced to decidualize in vitro and that this requires dbcAMP in addition to hormones. This is the first report demonstrating in vitro decidualization in a nonhuman primate.

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the <em>fibroblast</em> <em>growth</em> <em>factor</em> genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-<em>17</em> inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that <em>fibroblast</em> <em>growth</em> <em>factors</em> function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.