Three members of the connective tissue growth factor family CCN are differentially regulated by mechanical stress

Expression of connective tissue growth factor (CTGF), a member of the CCN gene family, is known to be significantly induced by mechanical stress. We have therefore investigated whether other members of the CCN gene family, including Cyr61 and Nov, might reveal a similar stress-dependent regulation. Fibroblasts growing under stressed conditions within a three-dimensional collagen gel showed at least a 15 times higher level of Cyr61 mRNA than cells growing under relaxed conditions. Upon relaxation, the decline of the Cyr61 mRNA to a lower level occurred within 2 h, and was thus quicker than the response of CTGF. The regulation was fully reversible when stress was reapplied. Thus, Cyr61 represents another typical example of a stress-responsive gene. The level of the Nov mRNA was low in the stressed state, but increased in the relaxed state. This CCN gene therefore shows an inverted regulation relative to that of Cyr61 and CTGF. Inhibition of protein kinases by means of staurosporine suppressed the stress-induced expression of Cyr61 and CTGF. Elevated levels of cAMP induced by forskolin mimicked the effects of relaxation on the regulation of Cyr61, CTGF and Nov. Thus, adenylate cyclase as well as one or several protein kinases might be involved in the mechanoregulation of these CCN genes.

Beta2-adrenergic receptor agonists reduce proliferation but not protein synthesis of periodontal fibroblasts stimulated with platelet-derived growth factor-BB

OBJECTIVE Catecholamines released from β-adrenergic neurons upon stress can interfere with periodontal regeneration. The cellular mechanisms, however, are unclear. Here, we assessed the effect of catecholamines on proliferation of periodontal fibroblasts. METHODS Fibroblasts from the gingiva and the periodontal ligament were exposed to agonists of the β-adrenergic receptors; isoproterenol (ISO, non-selective β-adrenergic agonist), salbutamol (SAL, selective β2-adrenergic receptor agonist) and BRL 37344 (BRL selective β3-receptor agonist). Proliferation was stimulated with platelet-derived growth factor-BB (PDGF-BB). Pharmacological inhibitors and gene expression analysis further revealed β-adrenergic signalling. RESULTS Gingiva and periodontal ligament fibroblast express the β2-adrenergic receptor. ISO and SAL but not BRL decreased proliferation of fibroblasts in the presence of PDGF-BB. The inhibitory effect of β-adrenergic signalling on proliferation but not protein synthesis in response to PDGF-BB was reduced by propranolol, a non-selective β-adrenergic antagonist. CONCLUSIONS These results suggest that β2-receptor agonists can reduce the mitogenic response of periodontal fibroblasts. These data add to the compelling concept that blocking of β2-receptor signalling can support tissue maintenance and regeneration.

Epidermal growth factor receptor (EGFR) is an independent adverse prognostic factor in esophageal adenocarcinoma patients treated with cisplatin-based neoadjuvant chemotherapy.

Neoadjuvant platin-based therapy is accepted as a standard therapy for advanced esophageal adenocarcinoma (EAC). Patients who respond have a better survival prognosis, but still a significant number of responder patients die from tumor recurrence. Molecular markers for prognosis in neoadjuvantly treated EAC patients have not been identified yet. We investigated the epidermal growth factor receptor (EGFR) in prognosis and chemotherapy resistance in these patients. Two EAC patient cohorts, either treated by neoadjuvant cisplatin-based chemotherapy followed by surgery (n=86) or by surgical resection (n=46) were analyzed for EGFR protein expression and gene copy number. Data were correlated with clinical and histopathological response, disease-free and overall survival. In case of EGFR overexpression, the prognosis for neoadjuvant chemotherapy responders was poor as in non-responders. Responders had a significantly better disease-free survival than non-responders only if EGFR expression level (p=0.0152) or copy number (p=0.0050) was low. Comparing neoadjuvantly treated patients and primary resection patients, tumors of non-responder patients more frequently exhibited EGFR overexpression, providing evidence that EGFR is a factor for indicating chemotherapy resistance. EGFR overexpression and gene copy number are independent adverse prognostic factors for neoadjuvant chemotherapy-treated EAC patients, particularly for responders. Furthermore, EGFR overexpression is involved in resistance to cisplatin-based neoadjuvant chemotherapy.

The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor.

INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic lung disease, resulting in respiratory insufficiency and reduced survival. Pulmonary fibrosis is a result of repeated alveolar epithelial microinjuries, followed by abnormal regeneration and repair processes in the lung. Recently, stem cells and their secretome have been investigated as a novel therapeutic approach in pulmonary fibrosis. We evaluated the potential of induced pluripotent stem cells (iPSC) conditioned media (iPSC-cm) to regenerate and repair the alveolar epithelium in vitro and improve bleomycin induced lung injury in vivo. METHODS IPSC-cm was collected from cultured iPSC derived from human foreskin fibroblasts and its biological effects on alveolar epithelial wound repair was studied in an alveolar wound healing assay in vitro. Furthermore, iPSC-cm was intratracheally instilled 7 days after bleomycin induced injury in the rat lungs and histologically and biochemically assessed 7 days after instillation. RESULTS iPSC-cm increased alveolar epithelial wound repair in vitro compared with medium control. Intratracheal instillation of iPSC-cm in bleomycin-injured lungs reduced the collagen content and improved lung fibrosis in the rat lung in vivo. Profibrotic TGFbeta1 and alpha-smooth muscle actin (alpha-sma) expression were markedly reduced in the iPSC-cm treated group compared with control. Antifibrotic hepatocyte growth factor (HGF) was detected in iPSC-cm in biologically relevant levels, and specific inhibition of HGF in iPSC-cm attenuated the antifibrotic effect of iPSC-cm, indicating a central role of HGF in iPSC-cm. CONCLUSION iPSC-cm increased alveolar epithelial wound repair in vitro and attenuated bleomycin induced fibrosis in vivo, partially due to the presence of HGF and may represent a promising novel, cell free therapeutic option against lung injury and fibrosis.

Treatment of exudative age-related macular degeneration with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study

PURPOSE To evaluate the safety, tolerability and bioactivity of ascending doses of MP0112, a designed ankyrin repeat protein (DARPin) that binds with high affinity to vascular endothelial growth factor-A (VEGF-A), in treatment-naive patients with exudative age-related macular degeneration (AMD). DESIGN Phase I/II, open-label, multicenter, dose-escalation study. METHODS Patients were to receive a single intravitreal injection of MP0112 at doses ranging from 0.04 to 3.6 mg and be monitored for 16 weeks for safety, efficacy, pharmacokinetics, and dose response. RESULTS Altogether, 32 patients received a single injection of MP0112. The maximum tolerated dose was 1.0 mg because of a case of endophthalmitis in the 2.0 mg cohort. Drug-related adverse events were reported by 13 (41%) of 32 patients; they included ocular inflammation in 11 patients (7 mild, 4 moderate in severity). Visual acuity scores were stable or improved compared with baseline for ≥4 weeks following injection; both retinal thickness and fluorescein angiography leakage decreased in a dose-dependent manner. Rescue therapy was administered to 20 (91%) of 22 patients who received 0.04-0.4 mg MP0112 compared with 4 of 10 (40%) patients who received 1.0 or 2.0 mg. Of patients in the higher-dose cohorts who did not require rescue treatment, 83% (5/6) maintained reductions in central retinal thickness through week 16. CONCLUSIONS A single injection of 1.0 or 2.0 mg MP0112 resulted in mean decreases in retinal thickness and leakage area despite ocular inflammation. Larger-scale studies are warranted to confirm these observations.

Scleral thinning after repeated intravitreal injections of antivascular endothelial growth factor agents in the same quadrant

PURPOSE We assessed the effects of intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy on scleral architecture using spectral domain anterior segment optical coherence tomography (OCT). METHODS A total of 35 eyes of 35 patients treated with at least 30 intravitreal injections in one eye in the inferotemporal quadrant with ranibizumab or aflibercept and 10 or less intravitreal injections in the fellow eye attending the intravitreal injection clinic were included. Enhanced depth imaging anterior segment OCT was used to measure scleral thickness. For each eye the sclera was measured in four quadrants at 3 mm from the limbus. In addition axial eye length was measured in all subjects using partial coherence interferometry. RESULTS The mean number of intravitreal injections was 42 (range, 30-73) and 1.6 (range, 0-9) in the fellow eyes. In the study eyes with more than 30 injections the average scleral thickness in the inferotemporal quadrant was 568.4 μm (SD ± 66 μm) and 590.6 μm (SD ± 75 μm) in the fellow eyes with 10 or less injections (P = 0.003). The mean average scleral thickness in the other three quadrants (inferonasal, superotemporal, and superonasal) was 536.6 μm in the study eyes (SD ± 100 μm) and 545.2 μm (SD ± 109 μm) in the fellow eyes (P = 0.22). There was a borderline association of the total number of injections with scleral thickness change in the inferotemporal quadrant (r = 0.3, P = 0.052). CONCLUSIONS Intravitreal injections may lead to scleral changes when applied repeatedly in the same quadrant. Thus, alternating the injection site should be considered in patients requiring multiple intravitreal injections.

Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I (IGF-I)

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.

Conditioned Medium of Demineralized Freeze-Dried Bone Activates Gene Expression in Periodontal Fibroblasts in Vitro.

BACKGROUND: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-β signaling in mesenchymal cells. The aim of the study was to determine whether processing of native bone into DBM affects the activity of the conditioned medium. METHODS: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium (DBCM). Changes in the expression of TGF-β target genes were determined. RESULTS: DBCM altered the expression of TGF-β target genes, e.g., adrenomedullin, pentraxin 3, KN Motif And Ankyrin Repeat Domains 4, interleukin 11, NADPH oxidase 4, and BTB (POZ) Domain Containing 11, by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-β receptor type I kinase inhibitor SB431542, but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-β target genes. CONCLUSION: The findings suggest that the conditioned medium from demineralized bone matrix can activate TGF-β signaling in oral fibroblasts. KEYWORDS: TGF-beta superfamily proteins; bone; bone substitutes; bone transplantation; conditioned media; freeze drying

Bevacizumab, Pemetrexed, and Cisplatin, or Bevacizumab and Erlotinib for Patients With Advanced Non-Small-Cell Lung Cancer Stratified by Epidermal Growth Factor Receptor Mutation: Phase II Trial SAKK19/09

Treatment allocation by epidermal growth factor receptor mutation status is a new standard in patients with metastatic nonesmall-cell lung cancer. Yet, relatively few modern chemotherapy trials were conducted in patients characterized by epidermal growth factor receptor wild type. We describe the results of a multicenter phase II trial, testing in parallel 2 novel combination therapies, predefined molecular markers, and tumor rebiopsy at progression. Objective: The goal was to demonstrate that tailored therapy, according to tumor histology and epidermal growth factor receptor (EGFR) mutation status, and the introduction of novel drug combinations in the treatment of advanced nonesmall-cell lung cancer are promising for further investigation. Methods: We conducted a multicenter phase II trial with mandatory EGFR testing and 2 strata. Patients with EGFR wild type received 4 cycles of bevacizumab, pemetrexed, and cisplatin, followed by maintenance with bevacizumab and pemetrexed until progression. Patients with EGFR mutations received bevacizumab and erlotinib until progression. Patients had computed tomography scans every 6 weeks and repeat biopsy at progression. The primary end point was progression-free survival (PFS) ≥ 35% at 6 months in stratum EGFR wild type; 77 patients were required to reach a power of 90% with an alpha of 5%. Secondary end points were median PFS, overall survival, best overall response rate (ORR), and tolerability. Further biomarkers and biopsy at progression were also evaluated. Results: A total of 77 evaluable patients with EGFR wild type received an average of 9 cycles (range, 1-25). PFS at 6 months was 45.5%, median PFS was 6.9 months, overall survival was 12.1 months, and ORR was 62%. Kirsten rat sarcoma oncogene mutations and circulating vascular endothelial growth factor negatively correlated with survival, but thymidylate synthase expression did not. A total of 20 patients with EGFR mutations received an average of 16.

L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

AIM To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. METHODOLOGY Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. RESULTS Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P < 0.05). This effect on VEGF was reduced by echinomycin (P < 0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P > 0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P < 0.05) and IL-8 levels (P < 0.05). CONCLUSIONS The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro

BACKGROUND Platelet-rich concentrates are used as a source of growth factors to improve the healing process. The diverse preparation protocols and the gaps in knowledge of their biological properties complicate the interpretation of clinical results. QUESTIONS/PURPOSES In this study we aimed to (1) analyze the concentration and kinetics of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF), leukocyte- and platelet-rich plasma (L-PRP), and natural blood clot during in vitro culture; (2) investigate the migration of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as a functional response to the factors released; and (3) uncover correlations between individual growth factors with the initial platelet/leukocyte counts or the induced cell migration. METHODS L-PRF, L-PRP, and natural blood clot prepared from 11 donors were cultured in vitro for 28 days and media supernatants collected after 8 hours and 1, 3, 7, 14, and 28 days. Released transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin growth factor (IGF-1), platelet-derived growth factor AB (PDGF-AB), and interleukin-1β (IL-1β) were measured in the supernatants with enzyme-linked immunosorbent assay. Migration of MSC and HUVEC induced by the supernatants was evaluated in Boyden chambers. RESULTS More TGF-ß1 was released (mean ± SD in pg/mL of blood) from L-PRF (37,796 ± 5492) compared with L-PRP (23,738 ± 6848; p < 0.001) and blood clot (3739 ± 4690; p < 0.001), whereas more VEGF and IL-1ß were released from blood clot (1933 ± 704 and 2053 ± 908, respectively) compared with both L-PRP (642 ± 208; p < 0.001 and 273 ± 386; p < 0.001, respectively) and L-PRF (852 ± 376; p < 0.001 and 65 ± 56, p < 0.001, respectively). No differences were observed in IGF-1 and PDGF-AB released from any of the concentrates. TGF-β1 release peaked at Day 7 in L-PRF and at 8 hours and Day 7 in L-PRP and 8 hours and Day 14 in blood clot. In all concentrates, main release of VEGF occurred between 3 and 7 days and of IL-1β between Days 1 and 7. IGF-1 and PDGF-AB were released until Day 1 in L-PRP and blood clot, in contrast to sustained release over the first 3 days in L-PRF. The strongest migration of MSC occurred in response to L-PRF, and more HUVEC migration was seen in L-PRF and blood clot compared with L-PRP. TGF-β1 correlated with initial platelet counts in L-PRF (Pearson r = 0.66, p = 0.0273) and initial leukocyte counts in L-PRP (Pearson r = 0.83, p = 0.0016). A positive correlation of IL-1β on migration of MSC and HUVEC was revealed (Pearson r = 0.16, p = 0.0208; Pearson r = 0.31, p < 0.001). CONCLUSIONS In comparison to L-PRP, L-PRF had higher amounts of released TGF-β1, a long-term release of growth factors, and stronger induction of cell migration. Future preclinical studies should confirm these data in a defined injury model. CLINICAL RELEVANCE By characterizing the biologic properties of different platelet concentrates in vitro, we may gain a better understanding of their clinical effects and develop guidelines for specific future applications.

First-Trimester Placental Growth Factor in Screening for Gestational Diabetes.

OBJECTIVE The aim of this study was first to assess whether first-trimester serum concentrations of placental growth factor (PlGF) differ between patients with and without gestational diabetes (GDM) and second to test whether there is a correlation between glycosylated hemoglobin (HbA1c), a factor recently shown to be useful in predicting GDM, and PlGF. METHODS PlGF was measured at 8-14 weeks with the Kryptor Immunoassay Analyzer (Brahms, Berlin, Germany). Absolute values were converted to multiples of the median using the software provided by the Fetal Medicine Foundation London. GDM was diagnosed using internationally accepted criteria. HbA1c levels were quantified using the TOSOH G7 automated hemoglobin analyzer. RESULTS From January to December 2014, 328 women were included in the study, 51 (15.5%) of whom developed GDM. First-trimester PlGF quantification does not discriminate between women at risk to develop GDM and controls, while HbA1c is able to do so. No correlation was found between PlGF and HbA1c. CONCLUSION Our findings do not lend support to the hypothesis that early PlGF values are different in women who later develop GDM.

Erratum to: Vascular endothelial growth factor induces contralesional corticobulbar plasticity and functional neurological recovery in the ischemic brain.

Erratum to: Acta Neuropathol (2012) 123:273–284. DOI 10.1007/s00401‑011‑0914‑z. The authors would like to correct Fig. 3 of the original manuscript, since the image in Fig. 3b does not correspond to a VEGF treated animal. Corrected Fig. 3 is shown below. We apologize for this mistake.

Placental expression of the angiogenic placental growth factor is stimulated by both aldosterone and simulated starvation

Aldosterone is an important factor supporting placental growth and fetal development. Recently, expression of placental growth factor (PlGF) has been observed in response to aldosterone exposure in different models of atherosclerosis. Thus, we hypothesized that aldosterone up-regulates growth-adaptive angiogenesis in pregnancy, via increased placental PlGF expression. We followed normotensive pregnant women (n = 24) throughout pregnancy and confirmed these results in a second independent first trimester cohort (n = 36). Urinary tetrahydroaldosterone was measured by gas chromatography-mass spectrometry and corrected for creatinine. Circulating PlGF concentrations were determined by ELISA. Additionally, cultured cell lines, adrenocortical H295R and choriocarcinoma BeWo cells, as well as primary human third trimester trophoblasts were tested in vitro. PlGF serum concentrations positively correlated with urinary tetrahydroaldosterone corrected for creatinine in these two independent cohorts. This observation was not due to PlGF, which did not induce aldosterone production in cultured H295R cells. On the other hand, PlGF expression was specifically enhanced by aldosterone in the presence of forskolin (p < 0.01) in trophoblasts. A pronounced stimulation of PlGF expression was observed with reduced glucose concentrations simulating starvation (p < 0.001). In conclusion, aldosterone stimulates placental PlGF production, enhancing its availability during human pregnancy, a response amplified by reduced glucose supply. Given the crucial role of PlGF in maintaining a healthy pregnancy, these data support a key role of aldosterone for a healthy pregnancy outcome.