Enhancing Dielectric Properties of (CaCu3Ti4O12 NWs-Graphene)/PVDF Ternary Oriented Composites by Hot Stretching.

Using high-dielectric inorganic ceramics as fillers can effectively increase the dielectric constant of polymer-based composites. However, a high percentage of fillers will inevitably lead to a decrease in the mechanical toughness of the composite materials. By introducing high aspect ratio copper calcium titanate (CaCu3Ti4O12) nanowires (CCTO NWs) and graphene as fillers, the ternary poly(vinylidene fluoride) (PVDF)-based composites (CCTO NWs-graphene)/PVDF with a significant one-dimensional orientation structure were prepared by hot stretching. CCTO NWs and graphene are arranged in a directional manner to form a large number of microcapacitor structures, which significantly improves the dielectric constant of the composites. When the ratio of CCTO NWs and graphene is 0.2 and 0.02, the oriented composites have the highest dielectric constant, which is 19.3% higher than the random composites, respectively. Numerical simulations reveal that the introduction of graphene and the construction of the one-dimensional oriented microstructure have a positive effect on improving the dielectric properties of the composites. This study provides a strategy to improve the dielectric properties of composite materials by structural design without changing the filler content, which has broad application prospects in the field of electronic devices.

Proteasome inhibitor attenuates skeletal muscle reperfusion injury by blocking the pathway of nuclear factor-kappaB activation.

BACKGROUND: Nuclear factor-kappaB is a key transcriptional factor in the regulation of inflammatory factors that are involved in tissue reperfusion injury, but conflicting data have been presented in the literature. The proteasome regulates proteins that control cell-cycle progression and apoptosis, and inhibition of the proteasome has been shown to reduce nuclear factor-kappaB activation and reperfusion injury. Although bortezomib is a potent proteasome inhibitor, its role in skeletal muscle reperfusion injury has not been documented, and its effects on the regulation of inflammatory factors in reperfused tissue are unclear. In this study, the authors investigated the role of nuclear factor-kappaB in skeletal muscle reperfusion injury and the effect of bortezomib (a proteasome inhibitor) on reperfusion injury. METHODS: Pedicled cremaster muscle flaps from bortezomib-treated and phosphate-buffered saline-treated control mice were subjected to 4.5 hours of ischemia and 90 minutes of reperfusion. RESULTS: During reperfusion, arterial diameters and blood flow recovered earlier and more completely in bortezomib-treated muscle than in controls. Compared with controls, Western blot analysis demonstrated a significant reduction in degradation of nuclear factor-kappaB inhibitory protein and expression of inducible nitric oxide synthase protein in bortezomib-treated muscle at the end of reperfusion. Immunohistochemistry showed decreased nuclear factor-kappaB p65-binding activity and down-regulated protein expression of intercellular adhesion molecule-1 and nitrotyrosine, accompanied by less muscle edema and inflammation as proven by histologic examination. CONCLUSIONS: Bortezomib effectively blocks nuclear factor-kappaB activation in attenuating muscle reperfusion injury through inhibiting nuclear factor-kappaB inhibitory protein degradation. Therefore, inhibition of proteasome activity may provide a novel therapeutic strategy for the treatment of skeletal muscle reperfusion injury.

Immunoproteasome down-modulation enhances the ability of dendritic cells to stimulate antitumor immunity.

The process of dendritic cell (DC) maturation, critical for effective DC-based immunotherapy, also alters the proteasome such that peptides presented in the context of HLA class I are generated not by the constitutive proteasome, but by the immunoproteasome. Cytotoxic T lymphocytes (CTLs) induced by such DCs might not optimally recognize tumor cells normally expressing the constitutive proteasome. Using small interfering RNA (siRNA) transfection of DCs to inhibit expression of the 3 inducible immunoproteasome subunits in mature DCs, we found that such DCs expressed increased intracellular levels of constitutive proteasomes and presented an altered repertoire of tumor-antigenic peptides. When DCs generated from the monocytes of 3 patients with melanoma were transfected with immunoproteasome siRNA, induced to mature, and then trans-fected with RNA encoding defined melanoma antigens, these DCs were superior inducers of antigen-specific CTLs against autologous melanoma cells. This alteration of DC proteasome composition, which enhances the ability of mature antigen-loaded DCs to stimulate anti-tumor immune responses, may lead to more effective DC-based tumor immunotherapy.

Intermittent pneumatic compression regulates expression of nitric oxide synthases in skeletal muscles.

This study investigated the effects of intermittent pneumatic compression (IPC) on expression of nitric oxide synthase (NOS) isoforms in compressed (anterior tibialis, AT) and uncompressed (cremaster muscles, CM) skeletal muscles. Following IPC application of 0.5, 1, and 5h on both legs of rats, the endothelial NOS (eNOS) mRNA expression was significantly up-regulated to 1.2-, 1.8, and 2.7-fold from normal, respectively, in both AT and CM, and protein expression increased more than 1.5-fold of normal at each time point. Similarly, neuronal NOS expression was up-regulated, but to a lesser degree. In contrast, inducible NOS expression was significantly and time-dependently down-regulated in both muscles. After IPC cessation, eNOS levels returned to normal in both AT and CM. The results confirm our hypothesis that IPC-induced vasodilation is mediated by regulating expression of NOS isoforms, in particular eNOS, in both compressed and uncompressed skeletal muscles. The results also suggest the importance of precisely characterizing expression of each NOS isoform in tissue pathophysiology.

Inhibition of iNOS attenuates skeletal muscle reperfusion injury in extracellular superoxide dismutase knockout mice.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are closely involved in the mechanism of skeletal muscle ischemia/reperfusion (I/R) injury. This study was designed to determine the effects of inducible nitric oxide synthase (iNOS) inhibitor 1400 W on the reperfused cremaster muscle in extracellular super-oxide dismutase knockout (EC-SOD(-/-)) mice. The muscle was exposed to 4.5 h of ischemia, followed by 90 min of reperfusion. Mice received either 3 mg/kg of 1400 W or the same amount of phosphate-buffered saline (PBS, as a control) subcutaneously at 10 min before the start of reperfusion. 1400 W treatment markedly improved the recovery speed of vessel diameter and blood flow in the reperfused cremaster muscle of EC-SOD(-/-) mice compared to controls. Histological examination showed reduced edema in the interstitial space and muscle fiber, and reduced density of nitrotyrosine (a marker of total peroxi-nitrate (ONOO(-)) level) in 1400 W-treated muscles compared to controls. Our results suggest that iNOS and ONOO(-) products are involved in skeletal muscle I/R injury. Reduced I/R injury by using selective inhibition of iNOS perhaps works by limiting cytotoxic ONOO(-) generation, a reaction product of nitric oxide (NO) and super-oxide anion (O(2) (-)). Thus, inhibition of iNOS appears to be a treatment strategy for reducing clinical I/R injury.

Inhibition of inducible nitric oxide synthase promotes recovery of motor function in rats after sciatic nerve ischemia and reperfusion.

PURPOSE: To investigate the effects of inhibition of inducible nitric oxide synthase (iNOS) on the recovery of motor function in the rat sciatic nerve after ischemia and reperfusion injury. METHODS: A 10-mm segment of the sciatic nerve from 169 rats had 2 hours of ischemia followed by up to 42 days of reperfusion. The animals were divided into 2 groups that received either iNOS inhibitor 1400W or the same volume of sterile water subcutaneously. A walking track test was used to evaluate the motor functional recovery during reperfusion. Statistical analysis was performed for the measurements of the sciatic functional index (SFI) by using 2-way analysis of variance; 1-way analysis of variance was used for the post hoc analysis of specific values at each time point of the SFI measurement. RESULTS: 1400W-treated rats had earlier motor functional recovery than controls, with a significantly improved SFI between days 11 and 28. Histology showed less axonal degeneration and earlier regeneration of nerve fibers in the 1400W group than in the controls. Inducible NOS messenger RNA and protein were up-regulated during the first 3 days of reperfusion but there was a down-regulation of neuronal NOS and up-regulation of endothelial NOS in control animals. 1400W treatment attenuated the increase of iNOS but had no effect on neuronal NOS and endothelial NOS. CONCLUSIONS: Our results indicate that early inhibition of iNOS appears to be critical for reducing or preventing ischemia and reperfusion injury.

The effects of exogenous nitric oxide donor on motor functional recovery of reperfused peripheral nerve.

PURPOSE: To investigate the effects of the nitric oxide donor S-nitroso-N-acetylcysteine (SNAC) on motor functional recovery of reperfused rat sciatic nerve. METHODS: Seventy-eight rats were divided into groups treated with SNAC (100 nmol/100 g/min), methylprednisolone 30 mg/kg/h for 15 minutes, 45-minute pause, 5.4 mg/kg/h for 1.5 h), and phosphate-buffered saline 0.2 mL/100 g/h). A 1-cm segment of sciatic nerve had 2 hours of ischemia and the results were evaluated after various reperfusion periods using a walking track test, muscle contractile testing, muscle weight, and histology. RESULTS: During reperfusion there was a significant overall improvement in sciatic functional index measurement and isometric titanic contractile force for the SNAC-treated group compared with the methylprednisolone- and phosphate-buffered saline- treated groups. The SNAC group had significantly earlier improvement in the sciatic functional index measurement between days 7 and 28. Restoration of the contractile force and muscle weight of the extensor digitorum longus muscle began earlier in the SNAC group--after day 11--whereas the other 2 groups showed progressive atrophy until day 21, with a significant difference between the SNAC group and the other 2 groups. Histologic examination showed that SNAC-treated rats had less severe degeneration and earlier regeneration of axons than the others. Although methylprednisolone-treated rats showed earlier recovery than phosphate-buffered saline-treated rats in all parameters there were no significant differences between these 2 groups. CONCLUSIONS: Supplementation of nitric oxide is effective in promoting motor functional recovery of the reperfused peripheral nerve and has potential to replace or augment steroids as therapeutic agents in treatment of nervous system ischemia/reperfusion injury.

Addition of nitric oxide donor S-nitroso-N-acetylcysteine to selective iNOS inhibitor 1400W further improves contractile function in reperfused skeletal muscle.

This study examines the effects of combination therapy with the nitric oxide (NO) donor S-nitroso-N-acetylcysteine (SNAC) and the iNOS inhibitor N-(3-(aminomethyl)benzyl) acetamidine (1400W) on contractile function in reperfused rat skeletal muscle. The right extensor digitorum longus (EDL) muscles of 104 rats were subjected to 3 h of ischemia followed by reperfusion times of 3 h, 24 h, and 7 days. For each time period, rats were further divided into sham operation, control, 1400W only, and 1400W plus SNAC groups. In vitro muscle contractile functional testing was performed in an organ chamber with electrical stimulation. The results showed that twitch and isometric tetanic forces were significantly improved in the 1400W-alone group compared to controls for 24 h and 7 days, but not 3 h of reperfusion. However, all three time periods of reperfusion showed that combination treatment of 1400W + SNAC significantly improved muscle contractile force compared to both control and 1400W-only groups. This corresponded to the decreased tissue necrosis and inflammation seen with combination therapy histologically. Our results demonstrate that combination treatment of 1400W + SNAC promotes functional recovery in reperfused skeletal muscle, supporting that manipulation of NO levels with a NO donor and an iNOS inhibitor is more beneficial than either treatment in isolation.

Skeletal muscle reperfusion injury is enhanced in extracellular superoxide dismutase knockout mouse.

This study investigates the role of extracellular SOD (EC-SOD), the major extracellular antioxidant enzyme, in skeletal muscle ischemia and reperfusion (I/R) injury. Pedicled cremaster muscle flaps from homozygous EC-SOD knockout (EC-SOD-/-) and wild-type (WT) mice were subjected to 4.5-h ischemia and 90-min reperfusion followed by functional and molecular analyses. Our results revealed that EC-SOD-/- mice showed significantly profound I/R injury compared with WT littermates. In particular, there was a delayed and incomplete recovery of arterial spasm and blood flow during reperfusion, and more severe acute inflammatory reaction and muscle damage were noted in EC-SOD-/- mice. After 90-min reperfusion, intracellular SOD [copper- and zinc-containing SOD (CuZn-SOD) and manganese-containing (Mn-SOD)] mRNA levels decreased similarly in both groups. EC-SOD mRNA levels increased in WT mice, whereas EC-SOD mRNA was undetectable, as expected, in EC-SOD-/- mice. In both groups of animals, CuZn-SOD protein levels decreased and Mn-SOD protein levels remained unchanged. EC-SOD protein levels decreased in WT mice. Histological analysis showed diffuse edema and inflammation around muscle fibers, which was more pronounced in EC-SOD-/- mice. In conclusion, our data suggest that EC-SOD plays an important role in the protection from skeletal muscle I/R injury caused by excessive generation of reactive oxygen species.

siRNA mediated inhibition of MMP-1 reduces invasive potential of a human chondrosarcoma cell line.

Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis. Expression of MMP-1 has been reported as a prognostic predictor of recurrence in human chondrosarcoma, and studies using human chondrosarcoma cell lines indicate that MMP-1 expression levels correlate with in vitro invasiveness. These observations suggest that MMP-1 activity has a central role in cell egress from the primary tumor at an early step in the metastatic cascade. In this study, siRNA was used to investigate whether knock down of the MMP-1 gene could be used to inhibit invasiveness in a human chondrosarcoma cell line. The inhibitory effect of siRNA on endogenous MMP-1 gene expression and protein synthesis was demonstrated via RT-PCR, Northern blotting, Western blotting, collagenase activity assay, and an in vitro cell migration assay. The siRNA inhibited MMP-1 expression specifically, since it did not affect the expression of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) nor other collagenases. Most importantly, the siRNA mediated reduction in MMP-1 expression correlated with a decreased ability of chondrosarcoma cells to invade a Type I collagen matrix. The reduction of invasive behavior demonstrated by human chondrosarcoma cells transfected with MMP-1 siRNA and the specificity of this inhibition supports the hypothesis that this metalloproteinase molecule is involved in initiation of chondrosarcoma metastasis.

The effects of N(omega)-propyl-L-arginine on reperfusion injury of skeletal muscle.

N(omega)-Propyl-L-arginine (NPA) is reported to be a highly selective inhibitor of neuronal nitric oxide synthase (nNOS). This in vivo study observed its role in ischemia/reperfusion (I/R) injury in rat skeletal muscle. Our results showed that NPA infusion significantly increased vessel diameters and blood flow in reperfused cremaster muscle, and slightly increased contractile function in reperfused extensor digitorum longus (EDL) muscle. In addition, NPA treatment slightly increased I/R-mediated downregulation of nNOS and eNOS mRNA and protein levels. Although NPA showed a beneficial role in I/R injury, our in vivo data do not support NPA as a selective nNOS inhibitor. Also, our data do not provide any insight into the mechanism of NPA. Thus, the in vivo mechanism of action of NPA needs to be further identified, and the role of nNOS in skeletal muscle I/R still remains to be determined.

Inhibition of iNOS with 1400W improves contractile function and alters nos gene and protein expression in reperfused skeletal muscle.

This study examined the effects of 1400W, an inhibitor of inducible nitric oxide (iNOS), on contractile function and iNOS expression in reperfused skeletal muscle. The right extensor digitorum longus (EDL) muscle of 104 rats underwent a sham operation or 3-h ischemia followed by 3-h or 24-h reperfusion (I/R). Rats received 3 mg/kg 1400W, 10 mg/kg 1400W, or water subcutaneously. Results showed that EDL contractile function in both 1400W-treated groups significantly outperformed the controls at 24-h but not at 3-h reperfusion. Although iNOS expression increased in all three I/R groups during reperfusion, a significantly smaller increase was found in 1400W-treated muscles after 3-h reperfusion, and more dramatically so after 24-h reperfusion. Our results indicate that inhibition of iNOS preserved the contractile function in reperfused skeletal muscle, perhaps via downregulating iNOS expression. Protection by 1400W at 24-h reperfusion suggests that the role of iNOS in exaggerating reperfusion injury is more prominent in the later stages of injury.

NF-kappaB p65 involves in reperfusion injury and iNOS gene regulation in skeletal muscle.

This study investigated the effects of inhibition of NF-kappaB activation on microcirculation and inducible NOS expression in reperfused rat cremaster muscle. The muscle from 16 rats underwent 5-h ischemia and 90-min reperfusion. Each rat received NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC, 150 mg/kg) or phosphate-buffered saline 15 min before reperfusion. Results showed that PDTC treatment had a significant overall increase in muscle blood flow during reperfusion. Blood flow more rapidly recovered to and over baseline in the PDTC-treated group than in controls, with a significant difference at 10-30 min and 70-90 min. Expression of iNOS mRNA had a 167-fold increase from normal in controls, but was significantly (P < 0.05) reduced to a 63-fold increase in PDTC-treated muscles. In addition, PDTC treatment significantly (P < 0.05) decreased a reperfusion-induced increase in activated NF-kappaB p65 and nuclear p65 protein. Our results suggest that NF-kappaB is involved in I/R injury and that inhibition of NF-kappaB p65 activation affords protection against I/R injury, perhaps via downregulating expression of iNOS transcription.

Nitric oxide involvement in reperfusion injury of denervated muscle.

PURPOSE: To investigate whether inhibition of inducible nitric oxide synthase (iNOS) improves microcirculation in denervated and reperfused skeletal muscle. METHODS: The cremaster muscles of 52 rats received iNOS inhibitor 1400W (3 mg/kg) or phosphate buffered saline (PBS) and underwent either 3 hours of ischemia and 1.5 hours of reperfusion or a sham operation. During reperfusion the vessel diameters were measured by using intravital videomicroscopy and overall muscle blood flow was measured with laser Doppler flowmetry. The expression of NOS messenger RNA (mRNA) and protein was determined by using real-time reverse-transcription polymerase chain reaction and Western blot, respectively. RESULTS: 1400W treatment significantly increased the mean blood flow of the reperfused muscle compared with controls, and this was associated with significantly less vasospasm in 10 to 20 microm, 21 to 40 microm, and 41 to 70 microm arterioles. The expression of iNOS mRNA and protein in controls increased 23-fold and 6-fold from normal, respectively, but was reduced to only a 2-fold increase in the 1400W-treated muscles. The ischemia/reperfusion (I/R)-induced decrease of endothelial NOS (eNOS) and neuronal NOS (nNOS) expression in controls was not significantly changed after 1400W treatment. CONCLUSIONS: Our data support a nitric oxide-mediated mechanism in reperfusion injury and show the importance of inhibition of iNOS in reducing reperfusion injury in denervated skeletal muscle. Our results suggest potential benefits via inhibition of iNOS to improve clinical outcomes not only for hand surgeons who work in the microsurgery field, but also for other physicians whose work involves ischemia/reperfusion injury.

Reperfusion injury in skeletal muscle is reduced in inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) participates in many pathological events, and selective inhibition of iNOS has been shown to reduce ischemia-reperfusion (I/R) injury in different tissues. To further confirm its role in this injury process, I/R injury was observed in denervated cremaster muscles of iNOS-deficient (iNOS-/-) and wild-type mice. After 3-h ischemia and 90-min reperfusion, blood flow in reperfused muscle was 80 +/- 8.5% (mean +/- SE) of baseline at 10-min reperfusion and completely returned to the preischemia baseline after 20 min in iNOS-/- mice. In contrast, blood flow was 32 +/- 7.4% at 10 min and increased to 60 +/- 20% of the baseline level at 90 min in wild-type mice (P < 0.001 vs. iNOS-/- mice at all time points). The increased muscle blood flow in iNOS-/- mice was associated with significantly less vasospasm in all three sizes of arterial vessel size categories. The weight ratio to the contralateral muscle not subjected to I/R was greater in wild-type mice (173 +/- 11%) than in iNOS-/- mice (117 +/- 3%; P < 0.01). Inflammation and neutrophil extravasation were also more severe in wild-type mice. Western blot analysis demonstrated an absence of iNOS protein band in iNOS-/- mice and upregulation of iNOS protein expression in wild-type mice. Our results confirm the importance of iNOS in I/R injury. Upregulated iNOS exacerbates I/R injury and appears to be a therapeutic target in protection of tissues against this type of injury.

Type II collagen modulates the composition of extracellular matrix synthesized by articular chondrocytes.

The articular cartilage extracellular matrix (ECM) interfaces with chondrocytes and influences many biological processes important to cartilage homeostasis and repair. The alginate bead culture system can be viewed as a model of cartilage repair in which the chondrocyte attempts to recreate the pericellular matrix while maintaining a differentiated phenotype. The purpose of this study was to evaluate the alteration in epitopes of proteoglycan and tenascin synthesized by chondrocytes in the presence of exogenous extracellular type II collagen. We evaluated the effects on four biomarkers associated with the creation of the denovo matrix using ELISA and immunohistochemistry: keratan sulfate epitope (5D4), 3B3(-) neoepitope of chondroitin-6- sulfate, 3B3(+) chondroitinase-generated epitope of chondroitin-6-sulfate, and tenascin-C expression. TGF-beta1 stimulated the production of 3B3(+), 5D4, and tenascin-C in a dose-dependent manner and decreased 3B3(-) levels. Following the addition of exogenous type II collagen, 3B3(-) increased and tenascin-C decreased but did not change the direction of TGF-beta1 effects. In contrast, 5D4 expression decreased in the presence of collagen II as TGF-beta1 increased to 10 ng/ml. Interestingly, the amount of 3B3(+) epitope was not affected by the incorporation of type II collagen. Immunohistochemistry found there was no significant difference in distribution of these biomarkers in the presence and absence of extracellular type II collagen incorporation. These results elucidate the subtle biochemical differences in ECM synthesized by chondrocytes in the presence of type II collagen and further characterize the role played by ECM in the TGF-beta1 regulation of the articular cartilage physiology.

C1-esterase inhibitor and a novel peptide inhibitor improve contractile function in reperfused skeletal muscle.

To determine the role of inhibition of complement activation in the contractile function of skeletal muscle ischemia-reperfusion (I/R) injury, the rat extensor digitorum longus (EDL) muscles underwent 3 h ischemia and received human C1-esterase inhibitor (C1-INH, 100 IU/kg), a synthetic C1q A chain peptide with a similar inhibitory effect on activated C1 (peptide, 5 mg/kg), or human serum albumin control. Results showed a significant overall increase in tetanic contractile forces of the reperfused EDL in both C1-INH and peptide groups compared to controls. Maximum improvement occurred with peptide treatment at 120-Hz stimulation, with an increase in force from 38 +/- 4% of normal in controls to 52 +/- 4% in peptide-treated rats. There were no significant differences between C1-INH and peptide groups. Plasma C3 and C4 activities were significantly increased in both treated groups, suggesting inhibition of complement activation. Our results suggest that complement activation is involved in I/R injury, and inhibition of complement activation may therefore represent a potential therapeutic approach to reducing or preventing I/R injury.

Reperfusion injury is reduced in skeletal muscle by inhibition of inducible nitric oxide synthase.

This study evaluated the effects of the selective inducible nitric oxide synthase (iNOS) inhibitor N-[3-(aminomethyl)benzyl]acetamidine (1400W) on the microcirculation in reperfused skeletal muscle. The cremaster muscles from 32 rats underwent 5 h of ischemia followed by 90 min of reperfusion. Rats received either 3 mg/kg 1400W or PBS subcutaneously before reperfusion. We found that blood flow in reperfused muscles was <45% of baseline in controls but sharply recovered to near baseline levels in 1400W-treated animals. There was a significant (P < 0.01 to P < 0.001) difference between the two groups at each time point throughout the 90 min of reperfusion. Vessel diameters remained <80% of baseline in controls during reperfusion, but recovered to the baseline level in the 1400W group by 20 min, and reached a maximum of 121 +/- 14% (mean +/- SD) of baseline in 10- to 20-micro m arterioles, 121 +/- 6% in 21- to 40-micro m arterioles, and 115 +/- 8% in 41- to 70-micro m arteries (P < 0.01 to P < 0.001). The muscle weight ratio between ischemia-reperfused (left) and non-ischemia-reperfused (right) cremaster muscles was 193 +/- 42% of normal in controls and 124 +/- 12% in the 1400W group (P < 0.001). Histology showed that neutrophil extravasation and edema were markedly reduced in 1400W-treated muscles compared with controls. We conclude that ischemia-reperfusion leads to increased generation of NO from iNOS in skeletal muscle and that the selective iNOS inhibitor 1400W reduces the negative effects of ischemia-reperfusion on vessel diameter and muscle blood flow. Thus 1400W may have therapeutic potential in treatment of ischemia-reperfusion injury.

Tenascin-C expression and distribution in cultured human chondrocytes and chondrosarcoma cells.

Tenascin-C (TNC) is an oligomeric glycoprotein of the extracellular matrix with several distinct isoforms variably expressed during embryogenesis, tumorogenesis, angiogenesis and wound healing. In the normal human adult, TNC is found in large concentrations in articular cartilage, suggesting tissue-specific function. The purpose of this study was to determine the specific in vitro TNC splicing patterns of articular chondrocytes and a human chondrosarcoma cell line. Cells were cultured in a three-dimensional bead system and TNC splice variant expression and distribution were examined with the use of Western blotting techniques, semi-quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. At both the transcriptional and post-translational levels, the chondrocytes were found to express significantly higher levels of the smaller 220 kDa isoform (P < 0.01), which was predominantly incorporated into the matrix. The splicing pattern of the malignant cells was characterized by a higher proportion of the larger 320 kDa isoform which was extruded into the media. In vivo studies are necessary to verify the expression of the large TNC isoform in chondrosarcoma and the production and integration of the smaller isoform in normal chondroid matrix. In addition, elucidation of the biologic functions of the two major TNC isoforms may lead to the development of novel diagnostic and therapeutic approaches to chondrosarcoma.

Role of nitric oxide in vasodilation in upstream muscle during intermittent pneumatic compression.

This study investigated the dosage effects of nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on intermittent pneumatic compression (IPC)-induced vasodilation in uncompressed upstream muscle and the effects of IPC on endothelial NOS (eNOS) expression in upstream muscle. After L-NMMA infusion, mean arterial pressure increased by 5% from baseline (99.5 +/- 18.7 mmHg; P < 0.05). Heart rate and respiratory rate were not significantly affected. One-hour IPC application on legs induced a 10% dilation from baseline in 10- to 20-microm arterioles and a 10-20% dilation in 21- to 40 microm arterioles and 41- to 70-microm arteries in uncompressed cremaster muscle. IPC-induced vasodilation was dose dependently reduced, abolished, or even reversed by concurrently infused L-NMMA. Moreover, expression of eNOS mRNA in uncompressed cremaster muscle was upregulated to 2 and 2.5 times normal at the end of 1- and 5-h IPC on legs, respectively, and the expression of eNOS protein was upregulated to 1.8 times normal. These increases returned to baseline level after cessation of IPC. The results suggest that eNOS plays an important role in regulating the microcirculation in upstream muscle during IPC.