Maspin nuclear localization is linked to favorable morphological features in pulmonary adenocarcinoma.

Maspin, a mammary homologue of Serine Protease Inhibitors, has been shown to inhibit tumor progression and metastasis. Recently, its biological functions have been linked to its subcellular localization. Specifically, a nuclear, opposed to a combined nuclear and cytoplasmic localization has been associated with increased survival in human malignancies, including non-small cell lung cancer (NSCLC). However, it is not known whether transformation affects maspin expression during lung carcinogenesis, and whether its subcellular localization correlates with the morphological features of NSCLC. To address these questions, we studied maspin expression in a model of transformation of bronchial epithelial cells and in resected NSCLC. We found that decreased maspin accompanied chemical transformation of normal immortalized bronchial epithelial cells BEAS 2B. Immunohistochemistry revealed maspin expression to be virtually universal in NSCLC, occurring in 72/77 Adenocarcinoma (ACa), and 46/46 squamous cell carcinoma (SqCCa). SqCCa showed almost exclusively a combined nuclear-cytosolic stain. In contrast, nuclear maspin, but not combined nuclear-cytoplasmic maspin significantly correlated with low histological grade, lower proliferative rate, absence of invasion, and negative p53 stain in ACa. These data support the hypothesis that nuclear localization of maspin may stratify subtypes of NSCLC with favorable clinical-pathological features.

Identification of the neuroprotective molecular region of pigment epithelium-derived factor and its binding sites on motor neurons.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) family, is a survival factor for various types of neurons. We studied the mechanisms by which human PEDF protects motor neurons from degeneration, with the goal of eventually conducting human clinical trials. We first searched for a molecular region of human PEDF essential to motor neuron protection. Using a spinal cord culture model of chronic glutamate toxicity, we show herein that a synthetic 44 mer peptide from an N-terminal region of the human PEDF molecule that lacks the homologous serpin-reactive region contains its full neuroprotective activity. We also investigated the presence and distribution of PEDF receptors in the spinal cord. Using a fluoresceinated PEDF probe, we show that spinal motor neurons contain specific binding sites for PEDF. Kinetics analyses using a radiolabeled PEDF probe demonstrate that purified rat motor neurons contain a single class of saturable and specific binding sites. This study indicates that a small peptide fragment of the human PEDF molecule could be engineered to contain all of its motor neuron protective activity, and that the neuroprotective action is likely to be mediated directly on motor neurons via a single class of PEDF receptors. The data support the pharmacotherapeutic potential of PEDF as a neuroprotectant in human motor neuron degeneration.

Phosphomimetic mutants of pigment epithelium-derived factor with enhanced anti-choroidal melanoma cell activity in vitro and in vivo.

PURPOSE: Currently, choroidal melanoma is chemoresistant and there is no routine adjuvant chemotherapy for it. We investigated whether pigment epithelium-derived factor (PEDF) and its triple phosphomimetic mutants could more efficiently suppress melanoma tumor growth and metastasis, as well as how the triple phosphomimetic mutants act as antitumor agents. METHODS: Phosphomimetic mutants of PEDF were constructed by site mutagenesis. Lentiviruses carrying wild type (WT) PEDF, S24E114E227A (EEA)-PEDF, and S24E114E227E (EEE)-PEDF were produced in 293 fast-growing, highly transfectable (FT) cells and used to infect human choroidal melanoma cell line (OCM-1). The growth, migration, invasion and metastasis abilities of OCM-1 cells expressing WT-PEDF, EEA-PEDF or EEE-PEDF were investigated in vitro and in vivo, while the underlying mechanism of PEDF phosphomimetic mutants were investigated via Western blotting. RESULTS: OCM-1 cells infected with lentiviruses carrying WT-PEDF, EEA-PEDF, and EEE-PEDF displayed reduced proliferation, migration and invasion abilities, and were more prone to apoptosis. Cell media containing WT-PEDF, EEA-PEDF, or EEE-PEDF protein inhibited the tube forming capacity of human umbilical vein endothelial cells (HUVEC) in vitro. OCM-1 cells expressing WT-PEDF, EEA-PEDF, or EEE-PEDF displayed significantly reduced tumor growth and metastasis in the melanoma xenograft of nude mice models, with the PEDF mutants displaying much stronger effects than the wild type. The antitumor effects of PEDF are associated with the inhibition of VEGF and nuclear factor kappa-B (NF-κB) expression, as well as further inhibition of Akt phosphorylation. CONCLUSIONS: The phosphomimetic mutants of PEDF showed enhanced anti-melanoma activity by directly affecting tumor cells and indirectly affecting angiogenesis. These findings encourage the development of PEDF mutants as innovative anticancer agents.

A phase I study of recombinant human C1 inhibitor in asymptomatic patients with hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a congenital disorder with recurrent attacks of localized swelling of submucosal tissue, subcutaneous tissue, or both caused by a deficiency of the plasma protein C1 inhibitor (C1 esterase inhibitor [C1INH]). OBJECTIVE: We sought to evaluate the effects of recombinant human C1INH (rhC1INH) isolated from the milk of transgenic rabbits in 12 asymptomatic patients with HAE. METHODS: rhC1INH was intravenously administered at doses of 6.25 to 100 U/kg on 2 occasions. RESULTS: rhC1INH appeared safe and was well tolerated. The course of functional C1INH in plasma showed a full initial recovery (dose-normalized maximum concentration of about 0.02 U/mL/U/kg) and a dose-dependent clearance of rhC1INH. After infusion of rhC1INH at 100 U/kg, a clearance of approximately 13 mL/min, a half-life of approximately 3 hours, and a volume of distribution of approximately 3 L were observed. Infusion at this dose led to functional C1INH levels in plasma of at least twice the normal level for about 2 hours and greater than 0.4 U/mL for about 9 hours. rhC1INH displayed dose-dependent biologic activity by increasing the C4 level, which was about 2-fold at 12 hours after rhC1INH at 100 U/kg, and decreasing levels of cleaved C4. CONCLUSION: The observed safety profile and biologic activity of rhC1INH warrants further clinical studies to assess its efficacy in treating HAE attacks.

In vivo catabolism of human kallikrein-binding protein and its complex with tissue kallikrein.

We recently identified and purified a novel human kallikrein-binding protein (HKBP) from human plasma. The HKBP forms a 92 kd sodium dodecyl sulfate-stable and heat-stable complex with tissue kallikrein. This study was undertaken to characterize the plasma clearance and tissue distribution of exogenously administered HKBP and its complex with tissue kallikrein. Human tissue kallikrein was first incubated with purified HKBP, and the high-molecular-weight complex was separated from unbound proteins on a high-pressure liquid chromatography gel filtration column. Tissue kallikrein, kallikrein-binding protein, and their complex were labeled with iodine-125 and then injected intravenously into Sprague-Dawley rats. The disappearance rates of trichloracetic acid-precipitable radioactivity from the circulation were determined. The clearance profile of HKBP shows a nonlinear pattern with an apparent half-life of 65 minutes (n = 4). The plasma clearance of HKBP complexed with kallikrein shows a similar profile but a shorter half-life of 33 minutes (n = 3). HKBP and its complex with kallikrein were mainly taken up by the liver but to a lesser degree by the kidney, lung, and other tissues. Labeled human kallikrein has an apparent half-life of 8 minutes (n = 4), and its clearance consists of a fast and a slow component. The data indicate that kallikrein-HKBP complex is cleared from the circulation two times faster than that of the binding protein alone and that it persists in the circulation four times longer than kallikrein alone. The results support the notion that more than one pathway exists for the metabolism of tissue kallikrein and that HKBP plays a role in modulating tissue kallikrein's bioavailability.

Biodistribution and pharmacokinetics of 131I-labeled LEX 032, a recombinant hybrid of antichymotrypsin.

Pharmacokinetic and biodistribution studies were conducted in rats on a novel serine protease inhibitor, LEX 032, that was radiolabeled with 131I by the Bolton-Hunter reagent. LEX 032, a genetically engineered recombinant human nonglycosylated serpin, has been shown to have antiinflammatory properties in a number of animal models of inflammation and reperfusion injury. When 131I-LEX 032 was injected intravenously, a rapid whole body clearance of radioactivity was seen. Blood clearance followed a similar pattern. Forty-eight hours postinjection, 2.00 +/- 0.65 of the administered dose remained in the body. Greater than 59% of the radio-activity was excreted in the urine within the first 24 hr. Little radioactivity was found in the feces. With the exception of the thyroid, no significant organ-related uptake was noted. Radioactivity in the liver peaked at 20 min postinjection, with 1.00 +/- 0.13% administered dose/g and approximately 10% administered dose in the whole liver. At 1 hr, uptake in the kidney (9.30 +/- 1.52% administered dose/g) was the highest among all tissues, except for the thyroid. Gamma camera images were consistent with the biodistribution pattern. Pharmacokinetics and biodistribution were not affected by the dose of LEX 032 and were quite different from those of glycosylated wild type antichymotrypsin. These data indicate that LEX 032 exhibits the pharmacokinetics expected of a nonglycosylated 45 kDa protein.

Conformational lability of vitronectin: induction of an antigenic change by alpha-thrombin-serpin complexes and by proteolytically modified thrombin.

We previously showed that the alpha-thrombin-antithrombin III complex causes antigenic change in vitronectin as monitored by the monoclonal anti-vitronectin antibody 8E6 (Tomasini & Mosher, 1988). We have extended these studies to other protease-serpin complexes and to gamma-thrombin, a proteolytic derivative of alpha-thrombin. In the presence of heparin, recognition of vitronectin by 8E6 was increased 64- or 52-fold by interaction with the complex of alpha-thrombin and heparin cofactor II or the Pittsburgh mutant (Met358----Arg) of alpha 1-protease inhibitor, respectively. This was comparable to the value obtained with the alpha-thrombin-antithrombin III complex. Factor Xa-serpin complexes were approximately 4-fold less effective than the corresponding thrombin complexes. alpha-Thrombin-serpin complexes but not Xa-serpin complexes formed disulfide-bonded complexes with vitronectin. Antigenic changes and disulfide-bonded complexes were not detected when trypsin- or chymotrypsin-serpin complexes were incubated with vitronectin. gamma-Thrombin caused 7- and 34-fold increases in recognition of vitronectin by MaVN 8E6 in the absence and presence of heparin, respectively. In contrast, alpha-thrombin by itself had no effect. The antigenic change induced by gamma-thrombin was maximal when gamma-thrombin and vitronectin were equimolar, was not dependent on cleavage of vitronectin, and was abolished by inhibition of gamma-thrombin with Phe-Pro-Arg-chloromethyl ketone but not with diisopropyl fluorophosphate. These data indicate that alpha-thrombin is the component in alpha-thrombin-serpin complexes that induces the antigenic change in vitronectin, probably via a region that is preferentially exposed in gamma-thrombin.

The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase--GW311616A a development candidate.

The discovery of a potent intracellular inhibitor of human neutrophil elastase which is orally active and has a long duration of action is described. The pharmacodynamic and pharmacokinetic properties of a trans-lactam development candidate, GW311616A, are described.

Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations.

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases.