Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization

IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of approximately 80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.     

A highly stable cambialistic-superoxide dismutase from Antrodia camphorata: expression in yeast and enzyme properties

A cDNA encoding a putative superoxide dismutase (SOD) was identified in expressed sequence tags of Antrodia camphorata, a medicinal mushroom found only in Taiwan. The deduced protein was aligned with Mn-SODs and Fe-SODs from other organisms, this SOD showed greater homology to Mn-SOD. Functional A. camphorata SOD protein was overexpressed in yeast and purified. The purified enzyme showed two active forms on a 12.5% native PAGE, a dimer and a monomer. The dimeric protein's half-life of deactivation at 80 degrees C was 7 min, and its thermal inactivation rate constant K(d) was 9.87 x 10(-2)min(-1). The enzyme was stable in a broad pH range from 5-11; in the presence of 0.4M imidazole and 2% SDS. The atomic absorption spectrometric assay showed that 1.0 atom of manganese/iron (9:1) was present in each SOD subunit. The high stability of the enzyme make it better suited than other cambialistic-SODs for use in cosmetics. The SOD also documents its future utility in developing anti-inflammatory agent and in the treatment of chronic diseases.     

Nitric oxide prevents apoptosis of human endothelial cells from high glucose exposure during early stage.

Hyperglycemia is a major cause of diabetic vascular disease. High glucose can induce reactive oxygen species (ROS) and nitric oxide (NO) generation, which can subsequently induce endothelial dysfunction. High glucose is also capable of triggering endothelial cell apoptosis. Little is known about the molecular mechanisms and the role of ROS and NO in high glucose-induced endothelial cell apoptosis. This study was designed to determine the involvement of ROS and NO in high glucose-induced endothelial cell apoptosis. Expression of endothelial nitric oxide synthase (eNOS) protein and apoptosis were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to control-level (5.5 mM) and high-level (33 mM) glucose at various periods (e.g., 2, 12, 24, 48 h). We also examined the effect of high glucose on H(2)O(2) production using flow cytometry. The results showed that eNOS protein expression was up-regulated by high glucose exposure for 2-6 h and gradually reduced after longer exposure in HUVECs. H(2)O(2) production and apoptosis, which can be reversed by vitamin C and NO donor (sodium nitroprusside), but enhanced by NOS inhibitor (N(G)-nitro-L-arginine methyl ether), were collated to a different time course (24-48 h) to HUVECs. These results provide the molecular basis for understanding that NO plays a protective role from apoptosis of HUVECs during the early stage (<24 h) of high glucose exposure, but in the late stage (>24 h), high glucose exposure leads to the imbalance of NO and ROS, resulting to the observed apoptosis. This may explain, at least in part, the impaired endothelial function and vascular complication of diabetic mellitus that would occur at late stages.     

Normal surface properties of phosphatidylglycerol-deficient surfactant from dog after acute lung injury

Lung surfactant was isolated from bronchoalveolar lavage of dogs during the late phase of recovery (15 days) from acute alveolar injury induced by subcutaneous injection of N-nitroso-N-methylurethane. This surfactant was compared with surfactant from control dogs in terms of in vitro surface properties, phospholipid composition and protein content, and those of its subfractions. Phospholipid composition and protein content were similar in the two groups, except that phosphatidylglycerol (PG) was markedly reduced and phosphatidylinositol (PI) was increased in the experimental group. In both, isopycnic densities of their subfractions in continuous sucrose density gradient were identical. The time course of surfactant adsorption was similar in both groups. Minimum surface tension (gamma min) was 4.1 +/- 1.5 dynes/cm in the experimental dogs and 3.8 +/- 1.3 dynes/cm in the controls. Surface compressibility (SC), stability index (SI), and dynamic respreadability (DR) of the surfactants from the two groups were nearly identical. When compared to an artificial surfactant composed of dipalmitoyl phosphatidylcholine (DPPC) and PG in 9:1 molar ratio a mixture of DPPC-PI 9:1 prepared identically showed similar gamma min, SC, SI, and DR, and a much higher surface adsorption rate. These results suggest that PG is not essential for normal in vitro surfactant function and that its role may be assumed by PI.     

Preparation of highly potent Naja naja atra (Formosan cobra) antivenin.

As the preparation of high titer antiserum against Naja naja atra venom is a time-consuming process, attempts were made to develop a immunization procedure for producing highly potent antiserums within a short period. Rabbits were immunized for 12 weeks with (1) whole venom as used routinely in our Institute, (II) whole venom adsorbed on carboxymethyl-cellulose (CMC) and mixed with Freund's complete adjuvant and (III) neurotoxin adsorbed on CMC and adjuvant mixture followed by CMC-whole venom-adjuvant mixture. The results showed that one ml of the antiserum prepared by method (III) could neutralize 445 LD50 of whole venom, in other words, its potency was 4 and 40 times higher than those prepared by methods (II) and (I), respectively.     

Cell culture and characterization of cross-linked poly(vinyl alcohol)-g-starch 3D scaffold for tissue engineering

The research goal of this experiment is chemically to cross-link poly(vinyl alcohol) (PVA) and starch to form a 3D scaffold that is effective water absorbent, has a stable structure, and supports cell growth. PVA and starch can be chemically cross-linked to form a PVA-g-starch 3D scaffold polymer, as observed by Fourier transform infrared spectroscopy (FTIR), with an absorbency of up to 800%. Tensile testing reveals that, as the amount of starch increases, the strength of the 3D scaffold strength reaches 4 × 10⁻² MPa. Scanning electron microscope (SEM) observations of the material reveal that the 3D scaffold is highly porous formed using a homogenizer at 500 rpm. In an enzymatic degradation, the 3D scaffold was degraded by various enzymes at a rate of up to approximately 30–60% in 28 days. In vitro tests revealed that cells proliferate and grow in the 3D scaffold material. Energy dispersive spectrometer (EDS) analysis further verified that the bio-compatibility of this scaffold.