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It is widely accepted that angiogenesis is required for tumor progression. Vascular endothelial growth factor (VEGF) is a key molecule for tumor angiogenesis; however, its expressional regulation is not well understood during all stages of tumorigenesis. Using cell lines and surgical specimens of human non–small cell lung cancers (NSCLCs), we here show that platelet-derived growth factor-AA (PDGF-AA) is an essential autocrine regulator for VEGF expression. To directly assess the expression of PDGF-AA–dependent VEGF and its roles in tumorigenesis, we stably transfected established cell lines with their antisense genes. In addition, the levels of PDGF-AA and VEGF expression in surgical sections were measured and compared with clinicopathologic findings such as tumor size and patient prognosis. PDGF-AA tightly regulated VEGF expression and had a greater effect on tumor size and patient prognosis than did VEGF in both cell lines and surgical sections. PDGF-AA expression was not seen in the atypical adenomatous hyperplasia at all, whereas VEGF was occasionally seen. Furthermore, the frequency of VEGF expression was higher in advanced NSCLCs than in precancerous lesions, which was tightly correspondent to the results for PDGF-AA. These results indicate that PDGF-AA is an important regulator of the frequency and level of VEGF expression during the transition from a precancerous lesion to advanced cancer. The PDGF-AA/VEGF axis, therefore, may be a ubiquitous autocrine system for enhancing angiogenic signals, and PDGF-AA, and its related pathways could be a more efficient target of antiangiogenic therapy for cancers than VEGF and its pathways.

Supplementary Figure S1 from Platelet-Derived Growth Factor-AA Is an Essential and Autocrine Regulator of Vascular Endothelial Growth Factor Expression in Non–Small Cell Lung Carcinomas

Summary In this paper, we present a solvent-assisted steam-assisted gravity drainage (SA-SAGD) experiment with multicomponent solvent (i.e., condensate) using a large physical model. The sandpack for the experiment had a porosity of 0.33 and a permeability of 5.6 darcies in the cylindrical pressure vessel that was 1.22 m in length and 0.425 m in internal diameter. The sandpack was initially saturated with 93% Athabasca bitumen and 7% deionized water. The main objective of this research was to study the in-situ thermal/compositional flow and produced bitumen properties in SA-SAGD with condensate. After the preheating of the sandpack for 24 hours, SA-SAGD with 2.8-mol% condensate was performed at 50 cm3/min (cold-water equivalent) at 3500 kPa for 3 days. The experimental data of production, injection, and temperature distribution were recorded. Also, 10 samples of produced oil were taken and analyzed for density and asphaltene content. The sandpack was excavated after the experiment to analyze the asphaltene content in the remaining oil at different locations. A numerical simulation model was calibrated based on the data of material balance and temperature distribution, and it was validated with properties of the produced and excavated samples. The simulation model used fluid models based on experimental data of viscosities, densities, and bubblepoints for four condensate/bitumen mixtures. Results showed that SA-SAGD was efficient in bitumen recovery with a cumulative steam-to-oil ratio (SOR) that was two to three times smaller than that in SAGD using the same physical model. Detailed analysis of the calibrated simulation model indicated that SA-SAGD enabled the steam chamber to expand more efficiently with a smaller amount of water throughput than SAGD. Volatile solvent components tended to remain in the chamber, and the condensed solvent components acted as a miscible carrier for bitumen components. The analysis further showed that the more efficient oil recovery in SA-SAGD occurred with predominantly cocurrent flow of oil and water near the chamber edge. SA-SAGD recovered a larger amount of asphaltene components (i.e., less in-situ upgrading) than SAGD likely because of its lower chamber temperature, shorter production period, and enhanced local displacement efficiency.

Introduction: Innovative imaging modality for preclinical diagnosis of aortic dissection (AD) has been awaited. Hypothesis: We previously reported that synchrotron radiation-based X-ray phase-contrast tomography (XPCT) imaging revealed densitometrical changes in the tunica media in the aortic wall (TM) of AD. To replicate XPCT findings in clinical practice, we investigated the application of high-resolution ultrasound in intraoperative epi-aortic scan (EAS) imaging. Methods: Twelve patients (normal aorta (5), chronic AD (3), acute type-A AD(4)) were selected. Before beginning cardiopulmonary bypass, the mid-ascending aorta was exposed and an EAS was conducted using EPIQ CVx and an eL18-4 transducer. After aortic repairs, the aortic samples of AD were fixed and applied XPCT, to compare ultrasonographic intensity (UI) of EAS and densitometric changes in XPCT in the same samples. Results: In normal aorta, UI remained unchanged throughout the TM (Figure-1A). In chronic AD, high linear changes in the UI in the middle of the TM was observed in the aorta that had not been dissected (yellow arrow in Figure-1B). In acute AD, high linear changes in the UI were clearly observed in the middle of TM, and aortic dissection was observed as an extension of those liner changes (Figure-1C). A line profile of the UI between the intima and the adventitia showed that the UI remained unchanged in normal aorta, and exhibited a high peak in the middle of the TM in chronic and acute AD (Figure-1A,B,C). These results indicated differences in the UI throughout the TM between normal and pathological aortas. These findings were well consistent with changes in the TM of patients with AD, which were obtained using XPCT (Figure-1D,E). Conclusions: High-resolution ultrasonographic imaging could replace XPCT findings in clinical settings and lead to significant improvements in the presymptomatic diagnosis of various aortic wall pathologies, including aortic dissection.

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry. Approach and Results: Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathologic intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell–dominant region, and the plasma-derived components (apolipoprotein B and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non–oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes. Conclusions: In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathologic intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages. Graphic Abstract: A graphic abstract is available for this article.

To investigate enhanced oil recovery processes, we constructed a molecular model of a live heavy crude oil (digital oil) and studied the crude oil properties at the reservoir temperature and a wide...

The Steam Assisted Gravity Drainage (SAGD) process has been used for in-situ thermal bitumen recovery in Canada. Nevertheless, there are several drawbacks that could impair the SAGD performance. One of the main drawbacks of SAGD is nontrivial consumption of energy to generate steam. To curtail the consumption of steam, many SAGD variants have been proposed. For example, Vapor Extraction (VAPEX) was proposed as a solvent-based cold production method and Solvent-Assisted SAGD (SA-SAGD) was proposed as a solvent-assisted hybrid method. In those solvent-based/assisted methods, the dissolution of solvents in bitumen enhances the in-situ oil mobility. The speed of the mixing between solvents and bitumen in solvent-based/assisted recovery processes is dictated by dispersive mass flux which is proportional to the transverse dispersion coefficient. Diffusion coefficient, tortuosity and transverse dispersivity should be quantified to characterize the transverse dispersion coefficient. In this research, the diffusion coefficients of solvents in Athabasca bitumen were determined with X-ray computed tomography (CT). A medical X-ray CT scanner was used to capture the diffusion phenomenon occurring between fresh liquid solvents and bitumen. The experiments were designed to reproduce the in-situ high pressure, high temperature condition (3.5 MPa, 135C) where solvent and bitumen actually interact at the vapor chamber edge in the SA-SAGD process. Using our new °experimental system, we successfully measured molecular diffusion coefficients at the condition of the vapor chamber edge for the first time. In conclusion, the binary diffusion coefficients between solvents and bitumen were successfully measured at the condition where the mixing of solvents and bitumen actually occurs in the SA-SAGD process. The characterized diffusion coefficients will be input parameters for the reservoir simulation and semi-analytical models.

Solvent Assisted-Steam Assisted Gravity Drainage (SA-SAGD) has been studied as a more efficient process for extracting bitumen from oil sands than the SAGD process. In the SA-SAGD process, solvent is injected with steam to decrease the viscosity of bitumen by dissolution of the condensed solvent. The dissolution of solvent causes a composition change of bitumen, which can lead to asphaltene precipitation. The effects of the asphaltene precipitation have been studied as part of a solvent-based recovery process such as Vapor Extraction (VAPEX). One of the advantages of the asphaltene precipitation is in-situ upgrading of the bitumen, whereas the disadvantage is that it causes a formation damage. To evaluate the effect of the asphaltene precipitation in the SA-SAGD process, it is essential to investigate the asphaltene precipitation under the conditions expected in the SA-SAGD process. However, it takes a lot of time to obtain sufficient data with a conventional method to quantify asphaltene precipitation under high-pressure/high- temperature (HP/HT) conditions. Therefore, the aim of this study is to develop an experimental procedure to evaluate the asphaltene precipitation with pressure/volume/temperature (PVT) apparatus in a reasonable time. The complex phenomenon at the edge of the chamber in the SA-SAGD process was simplified to a model of repetitions of mixing and drainage processes, and the experiment was configured in this manner. Solvent was added to a pre-diluted bitumen sample in a PVT cell. The supernatant liquid was sampled to analyze the asphaltene weight fraction remaining in the liquid phase and evaluate the asphaltene precipitation amount in the PVT cell. This process was repeated with increase in the solvent concentration. The asphaltene precipitation amount (APA) is calculated from the sample analysis data with recurrence relations under several assumptions. This procedure enables a wide range of APAs to be obtained from a mixture of bitumen and solvent in a single experiment, which enables sensitivity analysis under various conditions. In this research, the experiment was conducted under two different temperature conditions of 120°C and 150°C and the pressure was fixed at 3.5 MPa. The APA curves obtained from both experiments had almost the similar trend. Another important observation is that even the multi-component solvent (as used at the operation site) can still induce asphaltene precipitation under the HP/HT conditions expected in the SA-SAGD process.

We constructed a molecular model (digital oil model) for heavy crude oil based on analytical data. Crude oil was separated into four fractions: saturates, aromatics, resins, and asphaltenes (SARA). The digital oil was constructed as a mixture of representative molecules of saturates, aromatics, resins, and lost components (low boiling-point compounds vaporized during drying), while asphaltenes of ∼0.4 wt % in the crude oil being ignored. Representative molecules were generated by quantitative molecular representation (QMR), a technique that provides a set of molecules consistent with analytical data, such as elemental composition, average molecular mass, and the proportions of structural types of hydrogen and carbon atoms, as revealed by 1H and 13C nuclear magnetic resonance. To enable the QMR method to be applicable to saturates, we made two developments: the first was the generation of nonaromatic molecules by a new algorithm that can generate a more branched structure by separating the chain bonding in...

Background and aims Pathologic intimal thickening (PIT) is an important stage of atherosclerosis that leads to atheroma. The present study aimed to clarify the pathogenesis of PIT in humans. Methods Coronary arteries were obtained from 43 autopsy subjects aged 15–49 years. Non-atherosclerotic intima and atherosclerotic intimal lesions were classified into four groups, i.e. diffuse intimal thickening, fatty infiltration, fatty streak, and PIT, and the number and density of macrophages and smooth muscle cells (SMCs) were determined. Components of the lesions and proliferative and apoptotic activities of macrophages and SMCs were investigated by immunohistochemistry and TUNEL assay. Results Extracellular lipids accumulated mildly in the fatty infiltration and fatty streak, and abundantly in the PIT to form the lipid pool. The extracellular lipids co-localized with apolipoprotein B and fibrinogen. Macrophage foam cells accumulated in the fatty streak and PIT, but no TUNEL-positive macrophages were detected in any lesion. No significant difference in the number of SMCs was found between the four groups, but the density of SMCs decreased in the fatty streak and PIT. The decrease correlated with an increase in the number of macrophages, and the accumulation of extracellular lipids in the lipid pool. Neither Ki-67-positive nor TUNEL-positive SMCs were detected in any lesion. Conclusions In PIT in human atherosclerosis, the lipid pool is formed by infiltration and deposition of plasma-derived lipids. Intimal SMCs are dispersed in association with macrophage infiltration and lipid pool formation without undergoing significant proliferation or death.

Objectives: We previously reported excellent findings of X-ray phase-contrast tomography (PCX) for visualization of the formalin-fixed human aortic wall samples, and PCX enabled to demonstrate changes of tunica media in acute type A aortic dissection (AADA) . This study evaluates quantitative and dynamic measurements of fresh aortic wall samples of AADA with this modality. Methods: Fresh human aortic samples of the ascending aorta (n=7) were obtained during emergent aortic repair for AADA. Formalin-fixed human aortic walls of AADA (n=15) and normal aorta (n=15) were also investigated. PCX is approximately 1000 times more sensitive than absorption-contrast X-ray imaging and effective resolution of PCX is 11.7 μm. Quantitative and dynamic measurement has been developed to visualize changes in imaging of fresh aortic wall under various tensile force to simulate physiological condition, in which aortic wall is stretched according to blood pressure. Results: In normal aorta, quantitative measurement of density of the media was 1.095±0.003(g/cm3), and no different between intimal side (1.083±0.002) and adventitial side (1.085±0.003). On contrast, in formalin-fixed aorta of AADA, the medial density was 1.063±0.027, significantly lower than normal aorta (Figure-1), and different between intimal side and adventitial side (1.061±0.008 vs 1.081±0.011, respectively; p Conclusions: X-ray phase-contrast tomography was a strong modality to understand aortic structures and pathogenesis of acute type A aortic dissection.

Emerging evidence indicates that the tight communication between vascular endothelial cells and mural cells using platelet-derived growth factor (PDGF)-BB is essential for capillary stabilization during the angiogenic process. However, little is known about the related regulator that determines PDGF-BB expression. Using murine models of therapeutic neovascularization, we here show that a typical lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, is an essential regulator determining PDGF-BB expression for vascular stabilization via a paracrine mode of action. The blockade of VEGF type 3 receptor (VEGFR3) using neutralizing antibody AFL-4 abrogated FGF-2-mediated limb salvage and blood flow recovery in severely ischemic hindlimb. Interestingly, inhibition of VEGFR3 activity not only diminished lymphangiogenesis, but induced marked dilatation of capillary vessels, showing mural cell dissociation. In these mice, VEGF-C and PDGF-B were upregulated in the later phase after induced ischemia, on day 7, when exogenous FGF-2 expression had already declined, and blockade of VEGFR3 or PDGF-BB activities diminished PDGF-B or VEGF-C expression, respectively. These results clearly indicate that VEGF-C is a critical mediator, not only for lymphangiogenesis, but also for capillary stabilization, the essential molecular mechanism of communication between endothelial cells and mural cells during neovascularization.

In this paper, tensile strength and behavior of low-intensity UV light irradiated poly-p-phenylene benzobisoxazole (PBO) fiber were investigated in monofilament tests. The tensile tests of a monofilament were carried out at a gauge length of 12.5 mm and deformation rate of 0.5 mm/min. Irradiation time was set to 0h, 1h, 10h, 100h and 1,000h, while radiance was arranged to become 2, 4 and 8 W/m2. It was found that the tensile strength distribution of UV irradiated PBO fibers can be approximated to a normal distribution. Regardless of the degree of radiance, the tensile strength tends to decrease gradually with an increase in irradiation time. As radiance intensifies, however, corresponding curved lines move to lower positions, an indication of the dependency of the tensile strength on radiance. The relationship between radiation dosage and tensile strength converges on this one curved line irrespective of the degree of radiance. Therefore radiation dosage should be a valid parameter to measure the degradation of the strength of the PBO fibers exposed to UV light irradiation. In addition, it is found by SEM observation that there are distinct differences between the fracture surface image of UV non-irradiated fiber and that of irradiated fiber. Regardless of UV-irradiation, PBO fibers have split in the direction in which it is set. But the split part in UV-irradiated fiber is shorter than in the UV-non-irradiated fiber because UV-irradiated fiber has split vertically in portions.

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

In the isopropylation of biphenyl (BP) over a one-dimensional fourteen-membered ring (14-MR) CIT-5 zeolite with CFI topology, the selectivities for 4,4′-diisopropylbiphenyl (4,4′-DIPB) were 50–70% in the bulk and encapsulated products at moderate temperatures below 300 °C. However, they decreased at high temperatures such as 350 °C. These results mean that CIT-5 zeolites can preferentially exclude the transition state of the bulky DIPB isomers from their channels, thus resulting in the selective formation of 4,4′-DIPB. However, the isomerization of 4,4′-DIPB occurred on external and internal acid sites of the zeolite.

Alkylation, i.e. isopropylation, s-butylation, and t-butylation, of biphenyl (BP) was examined over fourteen-membered ring (14-MR) zeolites, CIT-5, UTD-1, and SSZ-53, in order to elucidate the relationships between structure of zeolites and bulkiness of alkylating agents on the shape-selective catalysis. CIT-5 zeolite (CFI) yielded 4,4'-diisopropyl-biphenyl (4,4'-DIPB) in the level of 50-60% in the isopropylation in the range of 150-300 °C. 2,2'-, 2,3'-, and 2,4'-DIPB (2,x'-DIPB) isomers were obtained as the predominant DIPB isomers at lower temperatures, and the formation of 3,4'-and 3,3'-DIPB isomers increased with an increase in the temperature. However, the selectivities were in the level of 10-15% for UTD-1 (DON) and SSZ-53 (SFH) zeolites in the range of 150-350°C. The s-butylation with 1-butene gave results similar to the isopropylation, although the selectivities for 4,4'-di-s-butylbiphenyl (4,4'-DSBB) were higher than those for 4,4'-DIPB at 250 °C: 80-85% for CFI, 40-50% for DON, and 30-40% for SFH. High selectivity for 4,4'-di-t-butylbiphenyl (4,4'-DTBB) was observed in the t-butylation at 250 °C: 95% for CFI, 90% for DON, and 80% for SFH. These differences are due to the spatial difference in their channels, and also due to bulkiness of alkylating agents, propene, 1-butene, and 2-methylpropene. The selectivity for 4,4'-dialkylbiphenyl (4,4'-DABP) was governed by the exclusion of the bulky DABP isomers at the transition state by steric restriction in the zeolite channels.

At SPring-8, synchrotron radiation-based X-ray phase-contrast tomography (PCXI) has been developed to measure the inner structures of biological soft tissue without destroying them. To resolve the three-dimensional (3D) morphology, we have applied PCXI to various cardiovascular tissue samples, including the thoracic aorta, ductus arteriosus, and cardiac conduction system. In the aortic walls, PCXI demonstrated differences in 3D structures of tunica media of aortic dissection. These findings correlated well with the irregularity of the structure of the media. In the surgically excised sample of coarctation of the aorta, PCXI showed 3D morphological changes in transition from the ductus arteriosus to the descending aorta. PCXI is also useful for examining abnormalities of the cardiac conduction system in congenital heart defects. Synchrotron radiation-based X-ray phase-contrast tomography has strong modality for analyzing 3D morphology and is useful for understanding the pathophysiology of various cardiovascular surgical pathologies.

Background. AUosensitization is a major risk factor for graft failure in clinical bone marrow transplantation, even with an human leukocyte antigen (HLA)-matched combination under radiation-based conditioning regimens. The critical components of immunological memory in donor bone marrow graft rejection in allosensitized hosts remain unclear at present. Methods. C57BL/6-recipient mice, which had been intraperitoneally injected with splenocytes from donor C3H mice on day -35 (sensitized recipients), had been lethally irradiated with 10-Gy whole-body irradiation and were intravenously injected with T-cell-depleted bone marrow cells (TCD-BMC) from C3H mice or third-party SJL mice. Results. Lethally irradiated recipient mice, which had been sensitized by donor splenocytes 5 weeks before the transplantation of TCD-BMC, completely rejected the donor-BMC in a donor-specific manner, whereas none of the nonsensitized recipient mice, all of which showed full allogeneic chimerism, rejected the donor TCD-BMC. Antibody-mediated T cell and/or Natural Killer (NK) cell depletion did not improve the ability of the sensitized recipients to overcome the rejection even when a megadose of TCD-BMC was administered to the sensitized recipients. Furthermore, BMC rejection occurred in sensitized B cell-deficient mice. In adoptive transfer experiments, naive mice, which received a transfer of purified T cells from sensitized mice, rejected the donor BMC, but not those from nonsensitized mice. Moreover, naive mice, which received a transfer of serum containing antidonor immunoglobulin, rejected the donor BMC. Conclusions. These findings suggest that alloreactive memory T cells and antidonor immunoglobulin independently function to reject donor BMC in sensitized recipients.

The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H-dependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

It is widely accepted that angiogenesis is required for tumor progression. Vascular endothelial growth factor (VEGF) is a key molecule for tumor angiogenesis; however, its expressional regulation is not well understood during all stages of tumorigenesis. Using cell lines and surgical specimens of human non–small cell lung cancers (NSCLCs), we here show that platelet-derived growth factor-AA (PDGF-AA) is an essential autocrine regulator for VEGF expression. To directly assess the expression of PDGF-AA–dependent VEGF and its roles in tumorigenesis, we stably transfected established cell lines with their antisense genes. In addition, the levels of PDGF-AA and VEGF expression in surgical sections were measured and compared with clinicopathologic findings such as tumor size and patient prognosis. PDGF-AA tightly regulated VEGF expression and had a greater effect on tumor size and patient prognosis than did VEGF in both cell lines and surgical sections. PDGF-AA expression was not seen in the atypical adenomatous hyperplasia at all, whereas VEGF was occasionally seen. Furthermore, the frequency of VEGF expression was higher in advanced NSCLCs than in precancerous lesions, which was tightly correspondent to the results for PDGF-AA. These results indicate that PDGF-AA is an important regulator of the frequency and level of VEGF expression during the transition from a precancerous lesion to advanced cancer. The PDGF-AA/VEGF axis, therefore, may be a ubiquitous autocrine system for enhancing angiogenic signals, and PDGF-AA, and its related pathways could be a more efficient target of antiangiogenic therapy for cancers than VEGF and its pathways.

Three kinds of catalysts of goethites supported on γ-Al 2 O 3 (Fe/γ-Al 2 O 3 ), SiO 2 -Al 2 O 3 (Fe/SiO 2 -Al 2 O 3 ) and SiO 2 (Fe/SiO 2 ) were investigated in terms of the catalytic activity of methane oxidation. The specific surface areas of these catalysts were larger in common than the area of goethite with no supports. The Fe/γ-Al 2 O 3 catalyst has the highest performance in the low-temperature activity of methane oxidation which started at 623 K and completed at 923 K. When the Fe-content in Fe/γ-Al 2 O 3 was increased, the formation of goethite was observed by the X-ray analyses and the activity of this catalyst increased up to 6 mol%. After the catalytic methane oxidation at 823 K, it was observed that goethite in Fe/γ-Al 2 O 3 transformed to hematite which has been known as an active iron oxide in methane oxidation. The activity of Fe/γ-Al 2 O 3 was enhanced by the addition of sodium up to the Na-content of 5 mol% although it descended above the content due to the decrease of the specific surface area of the catalyst.

Iron oxides were prepared by air oxidation of suspension obtained by mixing solutions of NaOH and FeSO4•7H2O. The ratio R was defined by mixing molar ratio of cations in the respective solutions (Na/Fe). Goethite (α-FeOOH) was mainly obtained in the range of R ≤ 1.9 (acidic region) and R ≥ 2.6 (basic region). Methane oxidation over the iron oxides started at as low temperature as [Formula: see text], at which the goethite changed to a hematite (α- Fe 2 O 3). The conversion ratio was low by the acidic goethite, in spite of its low crystallinity and high specific surface area. The basic goethite showed high activity for methane oxidation and the activity decreased grddually with increasing R. The low activity by the acidic goethite is attributable to the existence of some more percents of residual sulfate ion. And the sulfate ions disturbed the arrangement of oxide ions in the crystal structure of hematite.

Bronchiolitis obliterans (BO) after lung transplantation prevents a satisfactory prognosis, and recent studies suggested that interleukin-10 (IL-10) gene transfer to distant organs could inhibit BO in rodent models. Although delivery of the therapeutic gene to a local airway would be favored to minimize systemic effects, current limitations include lower gene transfer efficiency to airway epithelium. As recombinant Sendai virus (SeV) can produce dramatically efficient gene transfer to airway epithelium, we determined if SeV-mediated IL-10 gene transfer to the local airway would inhibit bronchial fibrous obliteration in murine tracheal allografts. Administration of cyclosporine A (CsA) significantly promoted not only recovery of the injured airway epithelium but also SeV-mediated IL-10 expression (CsA- versus CsA+ =228+/-78 versus 3627+/-1372 pg/graft with 5 x 10(7) pfu), thereby suggesting the requirement of epithelia for efficient gene transfer. Even at the highest expression, no significant leakage of IL-10 was evident in the systemic circulation, and the induction of interferon-gamma was completely diminished on day 7 by IL-10 gene transfer. As a result, luminal loss was significantly prevented in allografts treated with SeV-IL-10 (luminal opening, all control groups: 0% respectively, and SeV-IL-10 5 x 10(7) pfu: 25.7+/-10.5%), an effect that was enhanced by short-term CsA treatment (SeV-IL-10 5 x 10(7) pfu with CsA: 63.7+/-12.7%). We propose that SeV is a useful vector that can target airway epithelium to prevent BO avoiding putative systemic effect.

Purpose. To investigate the causal relationship between VEGF and tissue factor (TF) expression, and its intracellular signaling in the retinal capillary endothelium both in vitro and in vivo. Methods. TF mRNA and protein expression in cultured bovine retinal capillary endothelial cells (BRECs) were detected by RT-PCR and western blotting. The expression and subcellular localization of Egr-1 were analyzed by immunocytochemistry and western blotting. Involvement of p44/p42 MAPK pathway in this signaling was assessed using PD98059. Electrophoretic mobility shift assay (EMSA) was performed using human TF Egr-1/Sp-1 overlapping promoter region (–85 to –70). Decoy oligonucleotide was transfected into BRECs to clarify the critical transcription factor mediating VEGF-induced TF gene expression. To evaluate the importance of GC rich region in VEGF-induced TF protein expression in rat retinas, Mithramycin was intraperitoneally administered. Results. VEGF stimulated TF mRNA and protein expression in cultured BRECs, reaching maximal effect after 4 h and 10 h, respectively. VEGF activated transcription factor Egr-1 within 60 min. Inactivation of Egr-1 by PD98059 resulted in the prohibition of VEGF-induced TF gene expression. EMSA revealed the increment of Egr-1 binding with TF promoter region by displacing Sp1 after treatment with VEGF. Transfection of the Egr-1/Sp-1 overlapping decoy into BRECs inhibited VEGF-dependent TF gene expression. Mithramycin almost completely suppressed VEGF-induced TF protein expression in retinal capillary system in vivo (80%, p

ゲータイト(α-FeO(OH))を前駆物質として合成した酸化鉄を用いてメタン酸化活性試験を行った．ゲータイトは水酸化ナトリウム水溶液と硫酸鉄水溶液を混合した懸濁液の空気酸化により調製され，その熱分解により酸化鉄を得た．酸化鉄によるメタン酸化活性は硫酸イオンの存在で抑制されることがわかった．そこで，ゲータイトを硫酸に浸漬してその表面に硫酸イオン(SO42−)を存在させ，残存した硫酸イオンがゲータイトから調製された酸化鉄の結晶構造とメタン酸化活性に及ぼす影響について詳しく検討した．その結果，硫酸イオンは250 °C付近で起こるゲータイトからヘマタイト(α-Fe2O3)への相転移温度を約30 °C高温側へシフトさせることが明らかになった．また，この硫酸イオンは鉄原子に電子を供与して鉄の酸化力を減少させ，300 °Cから500 °Cの温度範囲においてメタン酸化活性を低下させ，かつメタンの部分酸化を引き起こす．さらに硫酸イオンによって，相転移して生成したヘマタイト中に多くの格子欠陥が存在し，(113l)(l : 0，1，2)面に代表される鉄原子の並びに関係する特定の面以外，すなわち酸素が関与する原子配列を乱すことがわかった．硫酸処理されたゲータイトから得られたヘマタイトを触媒として，550 °C以上でメタン酸化活性試験を行ったところ，硫酸処理なしのゲータイトから得られたヘマタイトよりも高活性であった．550 °C以上の温度においてヘマタイト表面の硫酸イオンは脱離するが，硫酸イオンによってもたらされたヘマタイトの格子欠陥は残り，この格子欠陥が高活性の要因であると考えられる．

Although the tumor suppressor p53 protein (P53) immunoreactivity and its gene (p53) mutation were reported to be significant prognostic indicators for human lung adenocarcinomas, little is known regarding the relationship between the heterogeneous distribution of P53 and its genetic status in each tumor focus and the clinicopathological significance. To determine how P53 is heterogeneously stabilized in patients, we compared P53 expression to both the p53 allelic mutation in exon 2 approximately 9 by polymerase chain reaction-single strand conformation polymorphism using microdissected DNA fractions, and the immunohistochemical MDM2 expression. Of the 48 positive to P53 in 118 lung adenocarcinomas examined, 10 with heterogeneous P53 expression were closely examined. The higher P53 expression foci in 7 of 10 cases were less differentiated, histologically in respective cases, and were frequently associated with fibrous stroma. Two had genetic mutations in exon 7 of the p53 gene in both the high and low P53 expression foci of cancer tissue indicating no apparent correlation between heterogeneous P53 expression and the occurrence of gene mutation. Immunohistochemical expression of MDM2 was significantly lower in high P53 expression areas (p < 0.05, the mean labeling indices of high and low P53 expression areas being 4.2 +/- 5.4% and 13.6 +/- 12.2%, respectively). In addition, among all the 118 cases examined, MDM2 expression was significantly suppressed in cases of p53 gene mutation, simultaneously with P53 overexpression, as compared with cases without both the p53 mutation and expression (p < 0.001). These findings suggest that the heterogeneous stabilization of P53 in human lung adenocarcinomas could be partly due to suppressed MDM2 expression. The overexpression of non-mutated P53 may afford a protective mechanism in human lung adenocarcinomas.

The angiogenic activity of two human salivary gland tumor cell lines, ACCS from adenoid cystic carcinoma and IT-2 from mucoepidermoid carcinoma, was examined by stimulating tube formation by bovine capillary endothelial cells (BCE). ACCS and IT-2 were cultured in 20 or 3% oxygen, representing normoxic and hypoxic conditions, respectively, and conditioned medium (CM) was obtained from each culture. The BCE tubes stimulated by hypoxic CM were 1.59 (ACCS) and 1.42 (IT-2) times longer than those stimulated by normoxic CM. The tube-forming activity of CM was inhibited by preincubation with either anti-vascular endothelial growth factor (VEGF) IgG or anti-basic fibroblast growth factor (bFGF) IgG, suggesting that both VEGF and bFGF with angiogenic activity were present in the CM. This was confirmed by ELISA, which also demonstrated increased concentrations of both proteins in the hypoxic CM. Northern blot analysis showed an increased VEGF mRNA level in both carcinoma cells with hypoxia, while hypoxia did not affect the bFGF mRNA level in either cell line. The results suggest that both VEGF and bFGF are major angiogenesis factors in salivary gland tumors, and hypoxia-induced angiogenesis results from upregulation of VEGF and increased release of bFGF.

Syntheses of the all-silica and the aluminosilicate versions of CIT-5 (International Zeolite Association Code: CFI) were studied. As structure-directing agent (SDA), N(16)-methylsparteinium (MeSPA+) cation was used. Under static conditions, combination of this SDA and lithium cations was considered to be a key factor for efficient synthesis of the zeolite. The synthesis vessel has been observed to play a significant role during the synthesis. Synthesis in a sealed quartz tube succeeded whereas that in a Teflon-lined stainless steel autoclave failed. CIT-5 with high purity was obtained inside the autoclave in the presence of hollow quartz tubes. When rotating the Teflon-lined autoclave, CIT-5 was obtained in the absence of the quartz tubes. With or without rotation, very high silica [Al]-CIT-5 was synthesized using fumed silica (Cab-O-Sil) and aluminum nitrate as silica source and aluminum source, respectively. With rotation, [Al]-CIT-5 with relatively lower Si/Al ratio was obtained by using highly dealuminated Y zeolite (HDY) as silica source and aluminum nitrate as aluminum source. In each aluminosilicate sample, only tetrahedral aluminum was observed. The particle size of the product was highly dependent on the synthesis method.

Neovascularization is well known to occur in human atherosclerotic plaques, proliferative retinopathies, and malignant neoplasias. However, its pathophysiologic roles and mechanisms still remain unclear. In this study, histochemical examination of atherosclerotic plaques showed that vascular endothelial cell growth factor (VEGF) was expressed by the smooth muscle cells and foamy macrophages in the atherosclerotic intimas. The number of VEGF-positive cells was positively correlated with the number of intimal blood vessels. Moreover, VEGF gene transfer into rabbit carotid arteries using the hemagglutinating virus of Japan (HVJ)-liposomes showed that VEGF overexpression could induce the angiomatoid proliferation. In diabetic rats, VEGF was overexpressed in diabetic retinas, and thus the local overexpression of VEGF seemed to play an important role in the development of blood-retinal barrier breakdown in simple diabetic retinopathy. These results indicated that the VEGF can act as a local and endogenous regulator of endothelial cell functions and that VEGF induces the neovascularization under pathophysiologic conditions. On the other hand, the transfer of a decoy for the cis-element in the promoter region of the angiogenic factors would be an effective method for regulating angiogenesis, because other angiogenic factors' expression promoted by such cis-element could be simultaneously suppressed. Therefore, this method may supply a useful therapeutic tool for the regulation of pathologic angiogenesis.

Abstract —Neovascularization is well known to occur in human atherosclerotic plaques; however, its pathophysiological roles, mechanisms, and stimuli in atherogenesis still remain unclear. In this study, 525 tissue blocks of coronary artery tissue obtained at autopsy from 48 patients ranging in age from 20 to 93 years old (mean±SD, 71±15 years) were immunohistochemically examined for vascular endothelial growth factor (VEGF) expression in the atherosclerotic intimas. The atherosclerotic lesions were histopathologically classified into types I through VI, as proposed by the American Heart Association Committee, and the numbers of intimal blood vessels and VEGF-positive cells were then morphometrically counted in sections that were immunohistochemically examined with anti-CD34 and human VEGF antibodies, respectively. The more the atherosclerotic lesion type advanced, the more often the lesion contained intimal blood vessels, which were expressed as percentages of the intimal section with intimal microvessels, viz, diffuse intimal thickening (DIT): 0% (0/111); type I, 31% (32/104); II, 42% (10/24); III, 66% (77/117); IV, 72% (48/67); V, 79% (70/89); and VI, 100% (13/13), P P

Tissue factor (TF) protein was overexpressed by macrophages and smooth muscle cells (SMCs) and deposited in the extracellular matrix of atherosclerotic intimas, probably resulting in enhanced procoagulant activity and the intimate participation in either thrombus formation or intimal fibrin deposition after the exposure of flowing blood and permeated fibrinogen to TF in atherosclerotic lesions. On the other hand, APO(a) was localized both in the stroma and within some macrophages. Fibrin deposition, which was more frequently detected in the matrix of advanced lesions than in that of early lesions, occasionally colocated with cell- and matrix-associated TF and APO(a) deposited in the matrix. These findings further support the hypothesis that the coagulation and fibrinolysis systems can play an essential role in the initiation and progression of atherosclerosis through fibrin deposition both in atherosclerotic plaques and on the arterial surface by neointimal hypercoagulability and a hypofibrinolytic state, which can also participate in SMC proliferation due to the decreased activation of TGF-beta by embedded and deposited APO(a). The clinical implications of these phenomena may thus contribute to future investigations in the prevention and treatment of atherosclerotic diseases.

• Background: Although the choroidal neovascularization (CNV) is a common pathologic feature of a number of different eye diseases, its pathological mechanisms have not been fully elucidated. We investigated the expression of vascular endothelial growth factor (VEGF) in CNV using an experimental primate model. • Method: CNV was induced by intense laser photocoagulation in four monkey eyes. Single eyes were enucleated at 1, 3, 7 or 14 days after photocoagulation and examined immunohistochemically for VEGF, macrophage antigen, von Willebrand factor and glial fibrillary acidic protein (GFAP). Expression of VEGF mRNA was examined byin situ hybridization. • Results: One day after photocoagulation, the normal structure of the outer portion of the retina and the inner portion of the choroid was destroyed. Three days after photocoagulation, choroidal vascular endothelial cells migrated into the subretinal space through the defect in Bruch's membrane. Increased expression of VEGF was detected in the accumulating macrophages, migrating retinal pigment epithelial (RPE) cells and Muller cells. Maximal expression of VEGF was observed between 3 and 7 days after wounding, and many newly formed vessels extended into the subretinal space 7–14 days after photocoagulation. • Conclusion: VEGF derived from RPE cells, macrophages and Muller cells may play a role in the formation of CNV.

We have previously isolated rabbit liver microsomal metalloendopeptidase (MEP) as a candidate for the processing enzyme of vitamin K-dependent plasma proteins. A cDNA coding for MEP has revealed that it is structurally related to metalloendopeptidase-24.15, which catalyzes the proteolytic processing of several bioactive peptides. In this study we examined the tissue distribution and subcellular localization of MEP by light and electron microscopic immunohistochemical methods, in addition to Northern blot analysis. Chicken polyclonal antibodies were raised by using synthetic peptides AG1 (Met31-Asn46) and AG3 (Asp537-Gly551) derived from the sequence of MEP. Both anti-AG1 and anti-AG3 antibodies reacted specifically with MEP, as judged by Western blotting and immunohistochemical methods. Both antibodies gave an identical staining distribution, which was localized on the luminal cell surfaces and in the cytoplasm of the following organs: liver, brain, lungs, kidneys, esophagus, stomach, duodenum, pancreas, placenta, epididymis, uterus, ovary, and oviduct. Northern blot analysis revealed that the expression of MEP mRNA is similar to its immunohistochemical distribution except in the heart. These results suggest that MEP may participate more closely in a degradation role in peptide metabolism in various tissues than in a processing role of the proprotein, like metalloendopeptidase-24.15.

Tissue factor pathway inhibitor (TFPI) plays a key role in modulating tissue factor-dependent blood coagulation. This study was done to determine not only the inhibitory effects of recombinant human TFPI (rTFPI) on thrombus formation in rat models with disseminated intravascular coagulation (DIC), but also to identify the distribution of exogenous TFPI in vivo . Disseminated intravascular coagulation was induced by administering a priming dose of carrageenan 10 mg/kg body weight and was followed 24 hours later by a provocative dose of lipopolysaccharide (LPS) 500 mg/kg body weight. The rTFPI was administered intravenously at a dose of either 1 or 4 mg/kg body weight immediately after LPS treatment. Exogenous rTFPI at a dose of 4 mg/ kg significantly inhibited the consumption of fibrinogen, platelets and factor Vila (P < .05) and also reduced the number of fibrin thrombi formed in the liver, lungs, kidneys, and spleen (P < .05), whereas rTFPI at a dose of 1 mg/kg had no significant inhibitory effect on these DIC parameters. Recombinant human rTFPI activity was rapidly cleared from the plasma; however, a significant amount of the inhibitor was still present in tissues even 3 to 6 hours after intravenous administration. Exogenous TFPI was mainly identified in Kupffer cells, macrophages, and on the microvascular endothelial lining of different organs. In the kidney, rTFPI was identified on both the abluminal surface of the renal tubules and the luminal surface of the proximal convoluted tubules. No rTFPI, however, was detected in the hepatocytes. Tissue factor was mainly expressed by monocytes/macrophages. These findings suggest that TFPI plays an important role in modulating TF-dependent thrombogenesis. The elucidation of the rTFPI distribution and interactions in vivo might thus provide valuable insight into its inhibitory mechanisms as well as its therapeutic implications in DIC.

Monkey Clara cell 10 kDa protein (CC10) was purified from monkey lung lavage. This protein showed an apparent molecular weight of about 10 kDa and 5 kDa under non-reducing and reducing conditions, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From the amino acid sequence data, monkey CC10 protein consisted of two identical 70-amino-acid polypeptide chains joined by two cystine residues, and possessed sequence identities of 78.6%, 52.9%, 52.9%, and 44.3% with human CC10, rat CC10 (PCB binding protein), rabbit uteroglobin, and mouse CC10, respectively. When monkey CC10 was compared with rabbit uteroglobin (progesterone binding protein), two polar residues of Tyr-21 and Thr-60, important for progesterone binding specificity, were substituted for Phe-21 and Met-60, and thus monkey CC10 may not have a binding capacity with progesterone. Monkey CC10 also possessed a surface homology with lipocortin I (anti-inflammatory peptide), thus suggesting that monkey CC10 plays a role in the anti-inflammatory process at the air-liquid interface over the bronchio-bronchiolar epithelium.

The histopathological characteristics of the newly formed blood vessels in the atherosclerotic intima of human coronary arteries examined three-dimensionally and light microscopically using serial sections were as follows : (1) the vascular density in atherosclerotic intimas significantly correlated with the degree of luminal stenosis, (2) the neovascularization was prominent in the intimas showing chronic inflammatory infiltrate, formation of granulation tissue or atheromatous change, while decreased in the extensively hyalinized and calcified intimas, and (3) the intimal vasculature originated mainly from the adventitia and partly from the proper coronary lumen. These findings support the hypothesis that the arteriointimal angiogenesis represents an essential and coincidental response to arterial injuries occurring in atherosclerotic process.In vivo introduction of human VEGF mRNA into rabbit carotid arteries using HVJ-liposome method induced the prominent angiomatoid proliferation of endothelial cells in thickened intima due to fibromuscular hyperplasia. Thus, the interventional suppression of intimal neovascularization by gene therapy may be an effective therapy for the restenosis following PTCA in the futural perspectives.