Development of a Survival Prognostic Model for Non-small Cell Lung Cancer.

Lung cancer is a leading cause of cancer-related death, and >80% of lung cancer diagnoses are non-small-cell lung cancer (NSCLC). However, when using current staging and prognostic indices, the prognosis can vary significantly. In the present study, we calculated a prognostic index for predicting overall survival (OS) in NSCLC patients. The data of 545 NSCLC patients were retrospectively reviewed. Univariate and multivariate Cox proportional hazards regression analyses were performed to evaluate the prognostic value of clinicopathological factors. Age (hazard ratio [HR] = 1.25, 95% confidence interval [CI] = 1.02-1.54), TNM stage (III, HR = 1.64, 95% CI = 1.08-2.48; IV, HR = 2.33, 95% CI = 1.48-3.69), lung lobectomy (HR = 1.96, 95% CI = 1.45-2.66), chemotherapy (HR = 1.42, 95% CI = 1.15-1.74), and pretreatment hemoglobin level (HR = 1.61, 95% CI = 1.28-2.02) were independent prognosticators. A prognostic index for NSCLC (PInscl, 0-6 points) was calculated based on age (≥65 years, 1 point), tumor-node-metastasis (TNM) stage (III, 1 point; IV, 2 points), lung lobectomy (no, 1 point), chemotherapy (no, 1 point), and pretreatment hemoglobin level (low, 1 point). In comparison with the "PInscl = 0" subgroup (survival time = 2.71 ± 1.86 years), the "PInscl = 2" subgroup (survival time = 1.86 ± 1.24 years), "PInscl = 3" subgroup (survival time = 1.45 ± 1.07 years), "PInscl = 4" subgroup (survival time = 1.17 ± 1.06 years), "PInscl = 5" subgroup (survival time = 0.81 ± 0.78 years), and "PInscl = 6" subgroup (survival time = 0.65 ± 0.56 years) exhibited significantly shorter survival times. Kaplan-Meier survival analysis showed that patients with higher PInscl scores had poorer OS than those with lower scores (log-rank test: χ2 = 155.82, P < 0.0001). The area under the curve of PInscl for predicting the 1-year OS was 0.73 (95 % CI = 0.69-0.77, P < 0.001), and the PInscl had a better diagnostic performance than the Karnofsky performance status or TNM stage (P < 0.01). In conclusion, the PInscl, which is calculated from age, TNM stage, lung lobectomy, chemotherapy, and pretreatment hemoglobin level, significantly predicted OS in NSCLC patients.

Impact of molecular weight in four-branched star vectors with narrow molecular weight distribution on gene delivery efficiency.

A series of star-shaped cationic polymers, termed star vectors (SVs), has been developed as effective nonviral gene delivery carriers. In this study, we separated SVs into several fractions having different molecular weights with very narrow molecular weight distributions in order to examine in detail the influence of the molecular weight of the SVs on the gene transfection efficiency. As a model compound for several types of SVs, 4-branched poly(N,N-dimethylaminopropyl acrylamide) having a molecular weight (M(n)) of approximately 35 kDa and polydispersity of 1.6 was prepared by iniferter-based radical polymerization. The SVs were separated using size-exclusion chromatography to obtain seven fractions having M(n) ranging from 27 kDa to 73 kDa with polydispersity ranging from 1.1 to 1.2. All the fractionated SVs have similar pH of 10.2-10.4 and were able to interact with and condense luciferase-encoding plasmid deoxyribonucleic acid (DNA) to yield SV/DNA polyplexes. A water-soluble tetrazolium-1 (WST) assay showed that all SVs had minimal cellular cytotoxicity under an N/P charge ratio of 10. The critical micellar concentration decreased with an increase in the M(n) of the fractionated SVs; however, the particle size of the polyplexes, exclusion activity of ethidium bromide, and zeta-potential of the polyplexes increased. An in vitro evaluation using COS-1 cells at an N/P ratio of 10 showed that transfection activity increased almost linearly with M(n). The highest transfection activity was obtained for SVs with the highest M(n) (73 kDa), which was over 7 times that for the SVs with the lowest M(n) (27 kDa), the nonfractionated original SV, or PEI standard. The transfection efficiency was more correlated with the amphiphilicity or hydrophobicity of the SVs and the surface potential and condensate density of the polyplexes than with the particle size.

Photoinduced cross-linking of star vector for improvement of gene transfer efficiency.

This study aimed to investigate the effect of cross-linking of a cationic nonviral gene carrier on gene expression. As a precursor for photo-cross-linking, a star-shaped, six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide) (six-branched star vector, SV), which was previously designed as a gene carrier, was synthesized by iniferter-based living radical polymerization. Upon UV irradiation, the number-average molecular weight (Mn) of the SV increased from ca. 28 kDa to ca. 32 kDa (irradiation time, 180 min) and ca. 46 kDa (240 min) with broadness of the polydispersity due to the coupling reaction between the polymer radicals generated at the terminal ends of each branch of the SVs, resulting in the preparation of cross-linked SVs (CSVs) without the use of any chemical cross-linking agents. Irrespective of cross-linking, all the SVs were able to interact with and condense luciferase-encoding plasmid DNA to yield relatively stable polymer/DNA complexes (polyplexes) of approximate diameter 150 nm with zeta-potential of ca. 20 mV. However, a transfection study using several types of cell lines, HeLa, Hep G2, 293, and COS-1, showed that by cross-linking of SVs the luciferase activity increased drastically. The activity with CSV (Mn=ca. 46 kDa) was increased by at least 1 order of magnitude in the original SV (Mn=ca. 28 kDa), which was several-fold that in the SV with the same molecular weight in all cells. In all SVs, no significant cellular cytotoxicity was observed even at a high charge ratio of 45. The SV-based gene transfection was significantly enhanced by the cross-linking of the SVs.

Pretreatment Hemoglobin Level Is an Independent Prognostic Factor in Patients with Lung Adenocarcinoma.

Background/Aim: Few studies have reported the prognostic value of pretreatment hemoglobin levels in patients with lung adenocarcinoma (LA). In the present study, we retrospectively reviewed 306 LA patients for their prognosis associated with the pretreatment hemoglobin levels. Methods: Person-years and case fatality rate (CFR) were calculated from May 2010 to June 2017. Hazard ratio (HR) and 95% confidence intervals (CIs) were estimated using the Cox proportional hazards regression analysis. Survival curves were generated using the Kaplan-Meier analysis. Results: Patients with low pretreatment hemoglobin (LPHb) levels had a higher CFR than did patients with normal pretreatment hemoglobin (NPHb) levels (HR = 1.48, 95% CI = 1.06-2.08, and P=0.023). Overall survival of NPHb patients was significantly higher than that of LPHb patients (P < 0.05). Conclusion: Low pretreatment hemoglobin level was demonstrated to be an independent biomarker for poor prognosis in patients with LA.

Pre-treatment hemoglobin levels are an independent prognostic factor in patients with non-small cell lung cancer.

To date, few studies have reported the prognostic value of pre-treatment hemoglobin levels in patients with non-small cell lung cancer (NSCLC). In the present study, 416 patients with NSCLC were retrospectively reviewed. Univariate Cox proportional hazards regression analysis demonstrated that patients with normal pre-treatment hemoglobin (NPHb) levels had a greater chance of surviving for longer period, than did patients with low pre-treatment hemoglobin (LPHb) levels (HR, 2.05; 95% CI, 1.63-2.57; P<0.001). After adjustment for age, sex, tumor-node-metastasis stage, Karnofsky performance status, lung lobectomy, chemotherapy and radiotherapy, multivariate Cox proportional hazards regression analysis revealed that LPHb was an independent predictor for the poor prognosis of patients with NSCLC (HR, 1.86; 95% CI, 1.47-2.36; P<0.001). Estimation of the cumulative survival revealed that the overall survival of NPHb patients was significantly higher than that for LBHb patients (P<0.05), independent of whether the patients had received lung lobectomy or chemotherapy treatments. In conclusion, low pre-treatment hemoglobin levels were demonstrated to be an independent biomarker for poor prognosis in patients with NSCLC.

Development of self-expandable covered stents.

We newly developed self-expandable covered stents by combining two of our original technologies. Of these, the first is the dip-coating covering method that was developed previously for balloon-expandable stents; the other is the newly developed self-expandable Nitinol stents, namely, Sendai stents. The three types of covered stents with the expansion diameter of 4.5, 5.0, or 6.0 mm thus obtained had a laser-processed microporous elastomeric cover film (pore diameter: 100 microm, interpore distance: 250 microm). Although the film was extremely thin (approximately 15 microm), the film could be expanded without causing any damage, the strut was completely embedded within the film, and the luminal surface of the film was smooth and flat. Mechanical properties such as ideal flexibility to follow the shapes of arteries were almost retained even after covering. As appropriate drugs, the blood-contacting inner and tissue-contacting outer surfaces of the film were differentially coated with argatroban for antithrombogenicity or FK506 for anti-inflammation, respectively. The preliminary in vivo study indicated that the covered stents mounted in the delivery catheter were navigated and placed to appropriate position in the arteries, and permissible neointimal thickening after 1-month implantation was observed similarly in noncovered stents.

Serine protease inhibitors inhibit superoxide release and adherence in human neutrophils stimulated by granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha.

Stimulation of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor-alpha (TNF) results in increased superoxide (O2-) release and adherence. O2- release and adherence are dependent on activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Possible participation of serine proteases in GM-CSF- or TNF-induced activation of human neutrophils was explored with various serine protease inhibitors, including phenylmethylsulfonyl fluoride, L-1-tosylamido-2-phenylethyl-chloromethyl ketone and N-alpha-p-tosyl-L-lysine-chloromethyl ketone. GM-CSF- or TNF-induced O2- release and adherence were inhibited in parallel by pretreatment of neutrophils with these inhibitors. On the other hand, GM-CSF- or TNF-induced phosphorylation of ERK and p38 MAPK was unaffected by these inhibitors at the concentrations effective for the inhibition of O2- release and adherence. These findings suggest that serine proteases are involved in GM-CSF- and TNF-induced O2- release and adherence in human neutrophils and that serine proteases function downstream or independently of the activation of ERK and p38 MAPK.

In vitro maturation of "biotube" vascular grafts induced by a 2-day pulsatile flow loading.

Autologous vascular tissues with a small diameter, "biotubes," were developed in vivo using a novel concept in regenerative medicine, "in-body tissue architecture technology." The effect of pulsatile flow in vitro was investigated on the structural and functional properties of the biotubes. Silicone rods (diameter, 3.0 mm; length, 35.0 mm), used as molds, were embedded into dorsal subcutaneous spaces of Wister rats. After 4 weeks, the autologous tubular tissues formed around the rods were harvested. Some tissues were incubated for 2 days under pulsatile flow simulating conditions in the human arteries with small caliber (wall shear stress (WSS), 15.5-77.3 dyn/cm(2); circumferential stress (CS), 0.6-4.5 x 10(5) dyn/cm(2)). Upon flow loading, the sparse, randomly oriented collagen fibers in the biotubes became dense and oriented in the regular circumferential direction. Compliances (beta values) of the control (ca. 30) and flow-loaded (ca. 20) biotubes were equivalent to that of the human coronary arteries and femoral arteries, respectively. Further, upon flow loading, the burst pressure significantly increased from ca. 1000 mmHg to ca. 1800 mmHg, along with the alpha-SMA-positive cell ratio. Pulsatile flow loading in vitro for 2 days could induce biotube maturation in terms of collagen structures and mechanical properties.

Evaluation of self-expandable, FK506-coated, covered stents in canine animal model.

This study aimed to evaluate whether drug coating of the recently developed covered SENDAI stents--self-expandable stents covered with segmented polyurethane (SPU) films--reduces neointimal thickening in animal model. FK506, which is one of the most effective immunosuppressants, was used. Bare stents; non-coated, covered stents; and FK506-coated, covered stents were placed bilaterally in the external iliac arteries of beagle dogs. After 1-month observation period, angiography did not show significant stent-induced stenosis. Histological evaluation revealed a completely endothelialized intravascular lumen and the absence of thrombus formation. The area of the intimal thickening induced by the FK506-coated stents was significantly smaller than that induced by the non-coated stents, whereas it was larger in the case of both the covered stents than that in the case of the bare stent. In conclusion, FK506 treatment of the self-expandable, covered stents was confirmed to effectively inhibit intimal thickening, although the SPU film used for covering functioned as a drug carrier in addition to a scaffold for intimal formation.

Enhancement of star vector-based gene delivery to endothelial cells by addition of RGD-peptide.

This study aimed to investigate the feasibility of using a cationic nonviral gene carrier in endothelial cells for enhancing gene expression by the addition of an integrin-binding RGD peptide. A 4-branched cationic polymer of poly( N,N-dimethylaminopropylacrylamide) (star vector), developed as a gene carrier, could complex with the luciferase-encoding plasmid DNA under a charge ratio of 5 (vector/pDNA) to form polymer/DNA complexes (polyplexes). The addition of the RGD-containing peptide (GRGDNP) to the polyplex solution led to a decrease in the zeta-potential from ca. +30 to +20 mV along with the reduction in the particle size from ca. 300 to 200 nm. Additionally, a marked inhibition of polyplex aggregation was observed, indicating the coating of the polyplex surface with RGD peptides. A transfection study on endothelial cells showed that the luciferase activity increased with the amount of RGD peptides added to the polyplexes and exhibited minimal cellular cytotoxicity. The transfection activity further increased when cyclic RGD peptides (RGDFV) were used; the activity with RGD peptide addition was approximately 8-fold compared to that without RGD peptide addition. Gene delivery to endothelial cells was significantly enhanced by only the addition of RGD peptides to star vector-based polyplexes.

Development of salmon collagen vascular graft: mechanical and biological properties and preliminary implantation study.

Elastic salmon collagen (SC) vascular grafts were prepared by incubating a mixture of acidic SC solution and a fibrillogenesis-inducing buffer containing a crosslinking agent [water-soluble carbodiimide (WSC)] in a tubular mold at 4 degrees C for 24 h and then at 60 degrees C for 5 min. Subsequently, re-crosslinking in ethanol solution containing WSC was performed. The dimension of the SC grafts was easily controlled by changing the size of the mold used. The compliance (stiffness parameter: beta) and burst strength of the SC grafts (internal diameter, 2 mm; length, 20 mm; and wall thickness, 0.75 mm) that were prepared for implantation were 18.2 and 1434 mmHg, respectively; both these values were comparable with those of native vessels. Upon placement in rat subcutaneous pouches, the SC grafts were gradually biodegraded with little inflammatory reaction. The SC grafts were preliminarily implanted in rat abdominal aortas by using specially designed vascular connecting system. This system was used because the graft exhibited easy tearing and thus inadequate suturability. There was neither aneurysm formation nor graft rupture, but mild thrombus formation was seen within the 4-week observation period. These grafts may be ideal for use in regenerative medicine because we believe that SC would be completely replaced with native vascular tissues after implantation, although further improvement in the mechanical properties of the graft is needed for anastomosis.

Development of in vivo tissue-engineered autologous tissue-covered stents (biocovered stents).

Biocovered stents, which are stents covered with autologous membranous tissues, were developed by applying a novel concept based on in vivo tissue engineering. Balloon-expandable stents crimped on silicone rods as a mold (diameter: 2 mm) were embedded into dorsal subcutaneous pouches in rabbits. After 1 month, the struts of the stents were fully encapsulated with membranous connective tissues formed around the silicone rods. Upon removing the silicone rod, stents covered with tubular connective tissues, in which the struts were completely impregnated, were obtained as biocovered stents. These tissues were composed mainly of collagen and fibroblasts and had a thickness of less than approximately 200 microm with an excellent high burst strength of approximately 1000 mmHg. The luminal surface of the tissues was extremely flat and smooth. The stents could be mounted on balloon catheters with a hand crimping tool and could be expanded by inflation with little damage to the tissues. It is anticipated that these novel stents may greatly enhance early normal vascular reconstruction with high reliability, thereby reducing the rate of in-stent restenosis.

Synthesis and in vitro evaluation of novel star-shaped block copolymers (blocked star vectors) for efficient gene delivery.

Novel 4-branched diblock copolymers consisting of cationic chains as an inner domain and nonionic chains as an outer domain were prepared by iniferter-based living radial polymerization and evaluated as a polymeric transfectant. The cationic polymerization of 3-(N,N-dimethylamino)propyl acrylamide (DMAPAAm) using 1,2,4,5-tetrakis( N,N-diethyldithiocarbamylmethyl)benzene as a 4-functional iniferter followed by the nonionic block polymerization of N,N-dimethylacrylamide (DMAAm) afforded 4-branched diblock copolymers with controlled compositions. By changing the solution or irradiation conditions, 4-branched PDMAPAAms with molecular weights of 10,000, 20,000, and 50,000 were synthesized. In addition, by graft polymerization, PDMAPAAm-PDMAAm blocked copolymers with copolymer composition (unit ratio of DMAAm/DMAPAAm) ranging from 0.18 to 1.0 for each cationic polymer were synthesized. All polymers were shown to interact with and condense plasmid DNA to yield polymer/DNA complexes (polyplexes). A transfection study on COS-1 cells showed that the polyplexes from block copolymers with cationic chain length of approximately 50,000 and a nonionic chain length of 30,000, which were approximately 200 nm in diameter and very stable in aqueous media, had the most efficient luciferase activity with minimal cellular cytotoxicity under a charge ratio of 20 (vector/pDNA). The PDMAPAAm-PDMAAm-blocked, star-shaped polymers are an attractive novel class of nonviral gene delivery systems.

Deposition transfection technology using a DNA complex with a thermoresponsive cationic star polymer.

A novel non-viral gene transfection method in which DNA complexes were kept in contact with a deposition surface (deposition transfection) was developed. We designed a novel aqueous thermoresponsive adsorbent material for DNA deposition, which was a star-shaped copolymer with 4-branched chains. Each chain is comprised of a cationic poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm) block (Mn: ca. 3000 g x mol(-1)), which formed an inner domain for DNA binding and a thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) block (Mn: ca. 6000 g x mol(-1)), which formed an outer domain for surface adsorption. Complex formation between the copolymer and the luciferase-encoding plasmid DNA occurred immediately upon simple mixing in an aqueous medium; polyplexes approximately 100 nm in size were formed. Because the lower critical solution temperature of the polyplexes was approximately 35 degrees C, they could deposit on the substrate by precipitation from an aqueous solution upon warming, which was confirmed by quartz crystal microbalance (QCM) method and water contact angle measurement. When COS-1 cells were cultured on the polyplex-deposited substrate in a culture medium, the luciferase activity observed was higher than that observed on a DNA-coated substrate with or without the cationic polymer before and after complete adhesion and by conventional solution transfection using the polyplexes. The activity was enhanced with an increase in the charge ratio (surfactant/pDNA) with permissible cellular cytotoxicity.