Neutralization of placental growth factor as a novel treatment option in diabetic retinopathy.

The current standard of care in clinical practice for diabetic retinopathy (DR), anti-vascular endothelial growth factor (VEGF) therapy, has shown a significant improvement in visual acuity. However, treatment response can be variable and might be associated with potential side effects. This study was designed to investigate inhibition of placental growth factor (PlGF) as a possible alternative therapy for DR. The effect of the anti-PlGF antibody (PL5D11D4) was preclinically evaluated in various animal models by investigating different DR hallmarks, including inflammation, neurodegeneration, vascular leakage and fibrosis. The in vivo efficacy was tested in diabetic streptozotocin (STZ) and Akimba models and in the laser induced choroidal neovascularization (CNV) mouse model. Intravitreal (IVT) administration of the anti-PlGF antibody was compared to anti-VEGFR-2 antibody (DC101), anti-VEGF antibody (B20), VEGF-Trap (aflibercept) and triamcinolone acetonide (TAAC). Vascular leakage was investigated in the mouse STZ model by fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) perfusion and in the Akimba model by fluorescein angiography (FA). Repeated IVT administration of the anti-PlGF antibody reduced vascular leakage, which was comparable to a single administration of VEGFR-2 inhibition in the mouse STZ model. PL5D11D4 treatment did not alter retinal ganglion cell (RGC) density, as demonstrated by Brn3a staining, whereas DC101 significantly reduced RGC number with 20%. Immunohistological stainings were performed to investigate inflammation (CD45, F4/80) and fibrosis (collagen type 1a). In the CNV model, IVT injection(s) of PL5D11D4 dose-dependently reduced inflammation and fibrosis, as compared to PBS treatment. Equimolar single administration of the anti-PlGF antibody and aflibercept (21 nM) and TAAC decreased leukocyte and macrophage infiltration with 50%, whereas DC101 and B20 (21 nM) had no effect on the inflammatory response. Similar results were observed in the mouse STZ model on the number of microglia and macrophages in the retina. Repeated administration of PL5D11D4 (21 nM) and TAAC similarly reduced fibrosis, while no effect was observed after equimolar DC101, B20 nor aflibercept administration (21 nM). In summary, the anti-PlGF antibody showed comparable efficacy as well-characterized VEGF-inhibitor on the process of vascular leakage, but differentiates itself by also reducing inflammation and fibrosis, without triggering a neurodegenerative response.

Placental growth factor increases regional myocardial blood flow and contractile function in chronic myocardial ischemia.

Placental growth factor (PlGF) has a distinct biological phenotype with a predominant proangiogenic role in disease without affecting quiescent vessels in healthy organs. We tested whether systemic administration of recombinant human (rh)PlGF improves regional myocardial blood flow (MBF) and systolic function recovery in a porcine chronic myocardial ischemia model. We implanted a flow-limiting stent in the proximal left anterior descending coronary artery and measured systemic hemodynamics, regional myocardial function using MRI, and blood flow using colored microspheres 4 wk later. Animals were then randomized in a blinded way to receive an infusion of rhPlGF (15 µg·kg(-1)·day(-1), n = 9) or PBS (control; n = 10) for 2 wk. At 8 wk, myocardial perfusion and function were reassessed. Infusion of rhPlGF transiently increased PlGF serum levels >30-fold (1,153 ± 180 vs. 33 ± 18 pg/ml at baseline, P < 0.001) without affecting systemic hemodynamics. From 4 to 8 wk, rhPlGF increased regional MBF from 0.46 ± 0.11 to 0.85 ± 0.16 ml·min(-1)·g(-1), with a concomitant increase in systolic wall thickening from 11 ± 3% to 26 ± 5% in the ischemic area. In control animals, no significant changes from 4 to 8 wk were observed (MBF: 0.45 ± 0.07 to 0.49 ± 0.08 ml·min(-1)·g(-1) and systolic wall thickening: 14 ± 4% to 18 ± 1%). rhPlGF-induced functional improvement was accompanied by increased myocardial neovascularization, enhanced glycogen utilization, and reduced oxidative stress and cardiomyocyte apoptosis in the ischemic zone. In conclusion, systemic rhPlGF infusion significantly enhances regional blood flow and contractile function of the chronic ischemic myocardium without adverse effects. PlGF protein infusion may represent an attractive therapeutic strategy to increase myocardial perfusion and energetics in chronic ischemic cardiomyopathy.

Enhanced Antitumor Efficacy of PhAc-ALGP-Dox, an Enzyme-Activated Doxorubicin Prodrug, in a Panel of THOP1-Expressing Patient-Derived Xenografts of Soft Tissue Sarcoma.

Despite poor response rates and dose-limiting cardiotoxicity, doxorubicin (doxo) remains the standard-of-care for patients with advanced soft tissue sarcoma. We evaluated the efficacy of two tetrapeptidic doxo prodrugs (PhAc-ALGP-Dox or CBR-049 and CBR-050) that are locally activated by enzymes expressed in the tumor environment, in ten sarcoma patient-derived xenografts. Xenograft models were selected based on expression of the main activating enzyme, i.e., thimet oligopeptidase (THOP1). Mice were either randomized to vehicle, doxo, CBR-049 and CBR-050 or control, doxo, aldoxorubicin (aldoxo) and CBR-049. Treatment efficacy was assessed by tumor volume measurement and histological assessment of ex-mouse tumors. CBR-049 showed significant tumor growth delay compared to control in all xenografts investigated and was superior compared to doxo in all but one. At the same time, CBR-049 showed comparable efficacy to aldoxo but the latter was found to have a complex safety profile in mice. CBR-050 demonstrated tumor growth delay compared to control in one xenograft but was not superior to doxo. For both experimental prodrugs, strong immunostaining for THOP1 was found to predict better antitumor efficacy. The prodrugs were well tolerated without any adverse events, even though molar doses were 17-fold higher than those administered and tolerated for doxo.

PhAc-ALGP-Dox, a Novel Anticancer Prodrug with Targeted Activation and Improved Therapeutic Index.

Clinical use of doxorubicin (Dox) is limited by cumulative myelo- and cardiotoxicity. This research focuses on the detailed characterization of PhAc-ALGP-Dox, a targeted tetrapeptide prodrug with a unique dual-step activation mechanism, designed to circumvent Dox-related toxicities and is ready for upcoming clinical investigation. Coupling Dox to a phosphonoacetyl (PhAc)-capped tetrapeptide forms the cell-impermeable, inactive compound, PhAc-ALGP-Dox. After extracellular cleavage by tumor-enriched thimet oligopeptidase-1 (THOP1), a cell-permeable but still biologically inactive dipeptide-conjugate is formed (GP-Dox), which is further processed intracellularly to Dox by fibroblast activation protein-alpha (FAPα) and/or dipeptidyl peptidase-4 (DPP4). In vitro, PhAc-ALGP-Dox is effective in various 2D- and 3D-cancer models, while showing improved safety toward normal epithelium, hematopoietic progenitors, and cardiomyocytes. In vivo, these results translate into a 10-fold higher tolerability and 5-fold greater retention of Dox in the tumor microenvironment compared with the parental drug. PhAc-ALGP-Dox demonstrates 63% to 96% tumor growth inhibition in preclinical models, an 8-fold improvement in efficacy in patient-derived xenograft (PDX) models, and reduced metastatic burden in a murine model of experimental lung metastasis, improving survival by 30%. The current findings highlight the potential clinical benefit of PhAc-ALGP-Dox, a targeted drug-conjugate with broad applicability, favorable tissue biodistribution, significantly improved tolerability, and tumor growth inhibition at primary and metastatic sites in numerous solid tumor models.

Involvement of thyroid hormones in chicken embryonic brain development.

Thyroid hormones (THs) play an important role in vertebrate brain development by stimulating and coordinating cell proliferation, migration and differentiation. Several TH-responsive genes involved in these processes have been identified, but the information is mainly derived from studies of late brain development, while relatively little is known about the more early stages, prior to the onset of embryonic TH secretion. We have chosen the chick embryo to investigate the role of THs in both late and early brain development. T(4) and T(3) are found in chicken brain from the earliest stages tested (day 4). Indirect clues for the involvement of T(3) in brain development are found in the ontogenetic expression profiles of proteins regulating its bioavailability and action, including TH transporters, deiodinases and TH-receptors. All of them are expressed in whole embryos tested on day 2 of incubation and in developing brain tested from day 4 onwards. Their distribution patterns vary over time and according to the brain area and cell type studied. Hypothyroidism induced during the second half of incubation disturbs cell migration in the cerebellum, providing more direct evidence for the requirement for THs during the later stages of brain development. Direct morphological proof for the requirement for THs during the first half of incubation is still missing, but microarray analysis of telencephalon shows a clearly divergent gene expression profile in hypothyroid embryos. In vivo knockdown of TH transporters and deiodinases in chick embryos cultured ex ovo provides an excellent tool to study the role of THs in early brain development in more detail.

Intravitreally Injected Fluid Dispersion: Importance of Injection Technique.

Purpose: The purpose of this study was to evaluate the dispersion of intravitreally injected solutions and investigate the influence of varying injection techniques. Methods: This was a prospective study using enucleated porcine eyes and ultra-high-resolution computed tomography (UHRCT) scanning to visualize iomeprol intravitreal dispersion. Sixty eyes were divided over 12 different groups according to the injection procedure: fast (2 seconds) or slow (10 seconds) injection speed and needle tip location (6- and 12-mm needle shaft insertion or premacular tip placement verified by indirect ophthalmoscopy). For each of these combinations, eyes were either injected with the combination of V20I (which is an analogue of ocriplasmin) and iomeprol or iomeprol alone. Distance to the macula and volume measurements were performed at 1, 2, 3, and 5 hours after injection. Results: The measured contrast bolus volume increases slowly over time to an average of 0.70 (P = 0.03), 1.04 (P = 0.006), and 0.79 (P = 0.0001) cm3 5 hours after the injection for the 6-mm needle shaft insertion, 12-mm needle shaft insertion, and premacular needle tip placement, respectively. The distance to the macular marker was significantly lower for premacular needle tip placement injections compared with 6- and 12-mm needle shaft insertion depths. Conclusions: Ultra-high-resolution computed tomography with three-dimensional reconstruction offers the possibility to study the dispersion of intravitreally injected solutions in a noninvasive manner. Intravitreal premacular solution delivery is possible with an indirect ophthalmoscope-guided injection technique and significantly reduces the time to reach the posterior pole in respect to 6- and 12-mm needle insertion depths. The speed of injection does not influence dispersion significantly.

Thyroid hormone deiodination in birds.

Because the avian thyroid gland secretes almost exclusively thyroxine (T4), the availability of receptor-active 3,3',5-triiodothyronine (T3) has to be regulated in the extrathyroidal tissues, essentially by deiodination. Like mammals and most other vertebrates, birds possess three types of iodothyronine deiodinases (D1, D2, and D3) that closely resemble their mammalian counterparts, as shown by biochemical characterization studies in several avian species and by cDNA cloning of the three enzymes in chicken. The tissue distribution of these deiodinases has been studied in detail in chicken at the level of activity and mRNA expression. More recently specific antibodies were used to study cellular localization at the protein level. The abundance and distribution of the different deiodinases shows substantial variation during embryonic development and postnatal life. Deiodination in birds is subject to regulation by hormones from several endocrine axes, including thyroid hormones, growth hormone and glucocorticoids. In addition, deiodination is also influenced by external parameters, such as nutrition, temperature, light and also a number of environmental pollutants. The balance between the outer and inner ring deiodination resulting from the impact of all these factors ultimately controls T3 availability.

Placental growth factor 2--A potential therapeutic strategy for chronic myocardial ischemia.

OBJECTIVES: We investigated whether sustained infusion of recombinant human placental growth factor-2 (rhPlGF-2) improves myocardial perfusion and left ventricular (LV) function in a porcine model of ischemic cardiomyopathy (ICM). METHODS: We induced myocardial ischemia using a flow-limiting stent in the LAD. Four weeks later, we randomized pigs with confirmed myocardial dysfunction to blinded rhPlGF-2 administration (PlGF2, 15 µg/kg/day, 14 days) or PBS (CON). At 8 weeks, we measured hemodynamics, contractile function and regional perfusion at rest and during stress using MRI and microspheres. We evaluated neovascularization post mortem. RESULTS: RhPlGF-2 administration increased PlGF serum levels more than 63-fold (83 3 ± 361 versus 11 ± 5 pg/ml CON, P<0.05) without adverse effects. After 4weeks, rhPlGF-2 significantly enhanced perfusion in the ischemic region at rest (0.83 ± 0.32 versus 0.58 ± 0.21 ml/min/g CON, P<0.05) and during hyperemia (1.50 ± 0.50 versus 1.02 ± 0.46 ml/min/g CON, P<0.05). Consequently, regional contractile function in rhPlGF-2-treated pigs improved at rest (37 ± 15% versus 23 ± 9% CON, P<0.05) and during high dose dobutamine stress (53 ± 31% versus 27 ± 16% CON, P<0.05). Enhanced perfusion translated into a greater improvement in LV ejection fraction and in preload-recruitable stroke work in rhPlGF-2-treated animals than in CON (52 ± 11 versus 41 ± 9%, and 76 ± 24 versus 41 ± 21 mmHg, respectively, P<0.05 for both), which was associated with significantly greater vascular density in the ischemic region. CONCLUSIONS: In chronic ICM, systemic rhPlGF-2 administration significantly enhances regional myocardial perfusion, contractile function at rest and during stress, and induces a prominent recovery of global cardiac function. PlGF-2 protein infusion is safe and may represent a promising therapy in chronic ICM.

Type I iodothyronine deiodinase in euthyroid and hypothyroid chicken cerebellum.

Immunocytochemistry using polyclonal anti-type I deiodinase (D1) led to the localization of D1 protein in the internal granule cells of the cerebellum in 1-day-old chicks, which was confirmed by the presence of in vitro D1 activity. Western blot analysis of hepatic and cerebellar extracts revealed a band of 27 kDa. In hypothyroid embryos D1 was expressed in both the internal and external granule cell layer and the signal diminished with more severe hypothyroidism, which is in agreement with the expected downregulation of D1 activity during hypothyroidism. In accordance with the protein data, hypothyroidism resulted in the downregulation of cerebellar D1 mRNA. Finally, histological stainings confirmed that the lack of staining in the external germinal layer of 1-day-old euthyroid chicks was due to the fact that migration of immature granule cells from the external towards the internal layer was completed at this stage while cell migration was retarded in hypothyroid animals.

The dioxin-like PCB 77 but not the ortho-substituted PCB 153 interferes with chicken embryo thyroid hormone homeostasis and delays hatching.

The effects of the dioxin-like polychlorinated biphenyl (PCB) 77 and the ortho-substituted PCB 153 on thyroid hormone availability were investigated during the last week of embryonic development in chicken. High doses of these PCBs (1microg PCB 77 and 20microg PCB 153) were injected into chicken eggs at day 4 of incubation. Blood and tissue samples were collected from day 14 of incubation until 1 day after hatching. We did not observe influences of PCB 153 on thyroid hormone (TH) levels. Treatment with PCB 77, on the other hand, decreased plasma total T(4) concentrations but increased hepatic T(4) levels at day 14 of incubation. Later in development, at stages near the process of hatching, severe decreases of T(4) and T(3) levels were observed in the PCB 77 group, both in plasma and tissues. PCB 77 severely reduced the TH peak that normally coincides with the stage of internal pipping. This reduction was accompanied by a considerable delay in the moment of hatching. We conclude that the dioxin-like PCB 77, but not the ortho-substituted PCB 153, can decrease TH availability towards the end of embryonic development and hence disturb the process of hatching.

cDNA cloning, functional expression, and characterization of chicken sulfotransferases belonging to the SULT1B and SULT1C families.

A search of the chicken expressed sequence tag (EST) database identified 2 cDNA clones that appeared to represent members of the SULT1B and SULT1C enzyme families. These cDNAs were fully sequenced and found to contain full-length inserts. Phylogenetic analysis of the derived amino acid sequences clearly placed them as the first members of the chicken SULT1B and SULT1C families, respectively, to be identified, and we propose they be named SULT1B1 and SULT1C1. (CHICK)SULT1B1 shares approximately 60% amino acid sequence identity with mammalian SULT1B enzymes, whereas the closest neighbor to (CHICK)SULT1C1 was the ortholog (RAT)SULT1C1, with 68% identity. We cloned these cDNAs into the bacterial expression vectors from the pET series. Transformed Escherichia coli cells strongly expressed the recombinant proteins. Purification of the recombinant enzymes from E. coli was accomplished by a three-step procedure involving ammonium sulfate precipitation, anion exchange chromatography, and affinity chromatography. The purified enzymes displayed subunit molecular weights of approximately 35,000Da on SDS-PAGE, as predicted, and were both able to sulfate a wide range of compounds, including xenobiotics and endogenous substrates such as iodothyronines. Detailed kinetic analysis showed SULT1C1 was more prolific in that it was able to sulfate dopamine, tyramine, and apomorphine, which SULT1B1 was not. 2-Bromophenol was the best substrate for both enzymes. We also raised antibodies against these proteins, which were able to detect the SULTs by ELISA, and which were able to strongly inhibit the recombinant enzymes. This is the first detailed characterization of sulfotransferases from the chicken, and it demonstrates that the avian and mammalian SULT1 enzymes are closely related in both structure and function.

Cold-induced enhancement of avian uncoupling protein expression, heat production, and triiodothyronine concentrations in broiler chicks.

The relationships among avian uncoupling protein (avUCP) mRNA expression, heat production, and thyroid hormone metabolism were investigated in 7-14-day-old broiler chicks (Gallus gallus) exposed to a low temperature (cold-exposed chicks, CE) or a thermoneutral temperature (TN). After 7 days of exposure, CE chicks exhibited higher heat production (+83%, P<0.01), avUCP mRNA expression (+20%, P<0.01), and circulating triiodothyronine (T(3)) levels (+104%, P=0.07) for non-statistically different body weights and feed intake between 3 and 7 days of exposure as compared to TN chicks. Plasma thyroxine (T(4)) concentration was clearly decreased in CE chicks (-33%, P=0.06). The lower hepatic inner-ring deiodination activity (-47%) and the higher renal outer-ring deiodination activity (+75%) measured in CE compared to TN chicks could partly account for their higher plasma T(3) concentrations. This study describes for the first time the induction of avUCP mRNA expression by low temperature in chickens, as it has been previously shown in ducklings, and supports the possible involvement of avUCP in avian thermogenesis.