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.

Renal microvascular assembly and repair: power and promise of molecular definition.

Developmental assembly of the renal microcirculation is a precise and coordinated process now accessible to experimental scrutiny. Although definition of the cellular and molecular determinants is incomplete, recent findings have reframed concepts and questions about the origins of vascular cells in the glomerulus and the molecules that direct cell recruitment, specialization and morphogenesis. New findings illustrate principles that may be applied to defining critical steps in microvascular repair following glomerular injury. Developmental assembly of endothelial, mesangial and epithelial cells into glomerular capillaries requires that a coordinated, temporally defined series of steps occur in an anatomically ordered sequence. Recent evidence shows that both vasculogenic and angiogenic processes participate. Local signals direct cell migration, proliferation, differentiation, cell-cell recognition, formation of intercellular connections, and morphogenesis. Growth factor receptor tyrosine kinases on vascular cells are important mediators of many of these events. Cultured cell systems have suggested that basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) promote endothelial cell proliferation, migration or morphogenesis, while genetic deletion experiments have defined an important role for PDGF beta receptors and platelet-derived growth factor (PDGF) B in glomerular development. Receptor tyrosine kinases that convey non-proliferative signals also contribute in kidney and other sites. The EphB1 receptor, one of a diverse class of Eph receptors implicated in neural cell targeting, directs renal endothelial migration, cell-cell recognition and assembly, and is expressed with its ligand in developing glomeruli. Endothelial TIE2 receptors bind angiopoietins (1 and 2), the products of adjacent supportive cells, to signals direct capillary maturation in a sequence that defines cooperative roles for cells of different lineages. Ultimately, definition of the cellular steps and molecular sequence that direct microvascular cell assembly promises to identify therapeutic targets for repair and adaptive remodeling of injured glomeruli.

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.

Biomarkers of pulmonary hypertension in patients with scleroderma: a case-control study.

INTRODUCTION Significant pulmonary vascular disease is a leading cause of death in patients with scleroderma, and early detection and early medical intervention are important, as they may delay disease progression and improve survival and quality of life. Although several biomarkers have been proposed, there remains a need to define a reliable biomarker of early pulmonary vascular disease and subsequent development of pulmonary hypertension (PH). The purpose of this study was to define potential biomarkers for clinically significant pulmonary vascular disease in patients with scleroderma. METHODS The circulating growth factors basic fibroblast growth factor, placental growth factor (PlGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor, and soluble VEGF receptor 1 (sFlt-1), as well as cytokines (interleukin [IL]-1β IL-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-13, tumor necrosis factor-α, and interferon-γ), were quantified in patients with scleroderma with PH (n = 37) or without PH (n = 40). In non-parametric unadjusted analyses, we examined associations of growth factor and cytokine levels with PH. In a subset of each group, a second set of earlier samples, drawn 3.0±1.6 years earlier, were assessed to determine the changes over time. RESULTS sFlt-1 (p = 0.02) and PlGF (p = 0.02) were higher in the PH than in the non-PH group. sFlt-1 (ρ = 0.3245; p = 0.01) positively correlated with right ventricular systolic pressure. Both PlGF (p = 0.03) and sFlt-1 (p = 0.04) positively correlated with the ratio of forced vital capacity to diffusing capacity for carbon monoxide (DLCO), and both inversely correlated with DLCO (p = 0.01). Both PlGF and sFlt-1 levels were stable over time in the control population. CONCLUSIONS Our study demonstrated clear associations between regulators of angiogenesis (sFlt-1 and PlGF) and measures of PH in scleroderma and that these growth factors are potential biomarkers for PH in patients with scleroderma. Larger longitudinal studies are required for validation of our results.

Impact of MET targeting on tumor-associated angiogenesis and growth of MET mutations-driven models of liver cancer

Deregulated expression of the MET receptor tyrosine kinase has been reported in up to 50% of patients with hepatocellular carcinoma, the most abundant form of liver cancers, and is associated with decreased survival. Consequently, MET is considered as a molecular target in this malignancy, whose progression is highly dependent on extensive angiogenesis. Here we studied the impact of MET small molecule inhibitors on angiogenesis-associated parameters and growth of xenograft liver models consisting of cells expressing MET-mutated variants M1268T and Y1248H, which exhibit constitutive kinase activity. We demonstrate that MET mutations expression is associated with significantly increased production of vascular endothelial growth factor, which is blocked by MET targeting only in cells expressing the M1268T inhibitor-sensitive but not in the Y1248H inhibitor-resistant variant. Decrease in vascular endothelial growth factor production is also associated with reduction of tyrosine phopshorylation of the vascular endothelial growth factor receptor 2 expressed on primary liver sinusoidal endothelial cells and with inhibition of vessel formation. Furthermore, MET inhibition demonstrated an efficient anti-tumor activity and considerable reduction in microvessel density only against the M1268T-derived intrahepatic tumors. Collectively, our data support the role of targeting MET-associated angiogenesis as a major biological determinant for liver tumor growth control.

The Phosphoinositide 3-Kinase p110α Isoform Regulates Leukemia Inhibitory Factor Receptor Expression via c-Myc and miR-125b to Promote Cell Proliferation in Medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K) pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi)-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α) was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH) subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.

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.

THE ROLE OF B CELLS IN THE T CELL DEPENDENT RELEASE OF HUMAN B CELL GROWTH FACTOR

Quiescent human B cells are postulated to go through activation and proliferation phases before undergoing differentiative phase for immunoglobulin secretion. The present studies address some of the aspects of activation and proliferation phase of normal human B cells. The definitions of signals responsible for B cell activation and proliferation resulted in the development of a highly specific, reproducible B cell growth factor (BCGF) assay. This BCGF bioassay utilizes activation by rabbit anti-human IgM-antibody. The functional specificity of this assay for measuring BCGF activity was demonstrated by the finding that target B cells proliferated but did not differentiate. The factor specificity was determined by specific absorption of BCGF by anti-IgM activated B cells. This assay was utilized for the studies of T-B cell collaboration and the essential function of monocytes in the production and/or release of B cell growth factor in a syngeneic in vitro system. It is apparent that highly purified T cells are poor producers of BCGF by themselves and require monocytes to secrete significant quantities of BCGF upon PHA stimulation. Macrophage soluble factor, Interleukin 1, is capable of replacing monocyte function for the release of BCGF by activated T cells. In our studies, B cells are incapable to function as accessory cells to replace monocyte function. Normal B cells are also not capable of producing BCGF under our experimental observations. However, the addition of these B cells at an optimum cell density (T:B ratio 1:1) doubles the monocyte dependent release of BCGF by syngeneic T cells. The augmentative role of B cells is expanded to understand the mechanism of BCGF release by T cells. It is observed from our studies that DR antigen of B cell surface is involved in the release of BCGF. The functional difference between DR of B cells and monocytes is observed as IL-1 could replace DR-treated monocytes whereas failed to replace DR-treated B cells for the release of BCGF by T cells. This functional difference may be attributed to the reported microheterogeneity in DR of B cells and monocytes. The addition of irradiated B cells increased the monocyte dependent T cell proliferation, suggesting the increase of T cell pool for BCGF release. In summary, the development of a biological assay specific for B cell growth factor led to the delineation of an interesting role of B cells in the release of its own growth factor by T cells. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^