Min-Xia Wang

Genotype dependent and cigarette specific effects on endothelial nitric oxide synthase gene expression and enzyme activity

We explored the interactive effects of endothelial nitric oxide synthase (eNOS) genotypes and cigarette smoking on protein levels and enzyme activity in 33 postpartum placentas. Whilst the eNOS protein levels were lower in the rare allele (0.48±0.11, n=9 vs. 1.05±0.10, n=24, P<0.01), the eNOS enzyme activity was about 7‐fold higher in the rare allele (4556.2±255.4 vs. 621.8±180.5 cpm/mg/min, P<0.01). Smokers had lower eNOS protein levels (1.07±0.09 vs. 0.50±0.19, P<0.05) in both alleles. It reduced the eNOS activities only in the rare allele (non‐smokers: 6143.8±251.2, n=5, smokers: 2968.5±259.4, n=4, 52% reduction, P<0.01). We conclude that associations between eNOS polymorphism and protein levels and enzyme activities are modifiable by smoking, the effects of smoking are dependent on the eNOS genotypes.

Apoptosis in rotator cuff tendonopathy.

The aim of this study was to investigate the involvement of apoptosis (programmed cell death) in the pathogenesis of rotator cuff disorders. The edges of torn supraspinatus rotator cuff tendons were collected from patients with rotator cuff tear (n = 25). Samples of the intra‐articular portion of subscapularis tendons were collected from patients without rotator cuff tear as control (n = 6). To minimize individual variance, we also collected six pairs of supraspinatus tendon and subscapularis tendon from six patients with rotator cuff tears. Apoptosis was detected by in situ DNA end labelling assay and DNA laddering assay. Immunohistochemical staining was performed to identify cells undergoing apoptosis. Control subscapularis tendon had normal morphology. Tendon from torn supraspinatus rotator cuff showed significant mucoid degeneration. Within the areas of degeneration, there were large numbers of apoptotic cells. The percentage of apoptotic cells in the degenerative rotator cuff (34%) was significantly higher than that in controls (13%) (p < 0.001). The excessive apoptosis detected in degenerative rotator cuff tissue was confirmed by DNA laddering assays. This is the first report of excessive apoptosis in degenerating rotator cuff tendon. Cells undergoing apoptosis in rotator cuff were mainly fibroblast‐like cells. These finding indicate that apoptosis may play an important role in the pathogenesis of rotator cuff degeneration.

Involvement of cytochrome c release and caspase-3 activation in the oxidative stress-induced apoptosis in human tendon fibroblasts

Our previous studies have demonstrated that oxidative stress and apoptosis are involved in human tendon degeneration. The objectives of our current study were to investigate the effect of oxidative stress on human tendon cell apoptosis, and to explore pathways by which tendon cell apoptosis was induced. In vitro oxidative stress was created by exposure of cultured human rotator cuff tendon cells to H(2)O(2). Apoptotic cells were assessed by Annexin V-FITC staining and necrotic cells by propidium iodide (PI) staining using flow cytometry. Cytochrome c and caspase-3 protein expression were detected by Western blotting. A mini-dialysis unit was employed to increase the protein concentration of the cytosolic fraction. Caspase-3 activity was determined by a colorimetric assay. Tendon cell apoptosis induced by H(2)O(2) was both dose and time dependent. Addition of H(2)O(2) resulted in the release of cytochrome c to the cytosol, and an increase of caspase-3 activity and the expression of caspase-3 subunit. The data suggest that oxidative stress-induced apoptosis in human tendon fibroblasts is mediated via pathway(s) that includes release of cytochrome c from mitochondria to the cytosol and activation of caspase-3.

APP expression, distribution and accumulation are altered by aluminum in a rodent model for Alzheimer's disease.

Up-regulated expression of amyloid precursor protein (APP) occurs early in the cascade of events that leads to amyloid plaque formation in the human brain. APP gene up-regulation, mediated by activated NF-kappaB, is a response to stress from nM concentrations of aluminum ions, aluminum-disregulated iron ions, reactive-oxygen species, cytokines, and physical trauma. We examined in vivo effects of aluminum on APP in aged rats, obtained from previously-reported longitudinal studies, that chronically ingested aluminum in amounts equivalent to total dietary aluminum levels that Americans routinely ingest. These rats exhibited two outcomes: one group remained cognitively-intact, scoring as well on a memory-discrimination task in old age as in middle age. The other developed cognitive deterioration, obtaining significantly lower mean performance scores in old age than in middle age and exhibiting abnormal behaviors associated with dementia. We compared the expression, distribution and accumulation of APP in hippocampal and cortical tissue of these two rat groups. Compared to results from cognitively-intact rats, hippocampal and cortical tissue from the cognitively-deteriorated rats showed elevated APP gene expression, significantly more dense APP deposits in cytoplasm of neural cells, and APP-immunoreactive neurites that were swollen and varicose. This study shows aluminum routinely derived from chronic oral ingestion, that gradually accumulates in brain regions important for memory-processing, is sufficient to increase APP levels in neural cells of those regions. Aluminum may thus launch the cascade that results in the formation of amyloid plaques in human brain.

Cell death and tendinopathy

Apoptosis and necrosis are presently recognized as the two major types of physiological and pathological cell death. Apoptosis is a tightly regulated cell deletion process that differs morphologically and biochemically from necrotic cell death. Tendinopathy is defined as a tendon injury that originates from intrinsic and extrinsic etiological factors. Excessive apoptosis has recently been described in degenerative tendon. The increased number of apoptotic tendon cells in degenerative tendon tissue could affect the rate of collagen synthesis and repair. Impaired or dysfunctional protein synthesis may lead to weaker tendon tissue and eventually increase the risk for tendon rupture. Clearly, there are many details to insert into this pathway, but there is hope that if the fine details of the pathway can be fleshed out, then strategies may be able to be developed to break the cycle at one or more points and prevent or treat tendinopathy more effectively.

Decreased Renal Expression of Nitric Oxide Synthase Isoforms in Adrenocorticotropin-Induced and Corticosterone-Induced Hypertension

Abstract—Administration of adrenocorticotropic hormone (ACTH) leads to the development of hypertension. Because glucocorticoids can affect the nitric oxide system at several sites, the present study tested the hypothesis that nitric oxide synthase (NOS) expression may be altered in ACTH-induced and corticosterone-induced hypertension in the rat. This was addressed by measuring Nos1, Nos2, and Nos3 mRNA in the kidney, adrenal gland, heart, and hypothalamus of 16 ACTH-treated and 16 vehicle-treated rats as well as in 10 corticosterone-treated and 10 control rats. In addition, in situ hybridization and immunohistochemistry were used to confirm changes by detection of Nos in RNA and NOS protein in tissues. Systolic blood pressure of ACTH and corticosterone rats was elevated (165±6 and 162±11 mmHg;P <0.001 versus control). Each Nos isoform mRNA was measured by reverse transcriptase-polymerase chain reaction technique. In ACTH rats, mRNA for Nos2 was reduced in renal cortex by 58±5% and in medulla by 68±7%; for Nos3, mRNA reductions of 59±6% and 51±11% were seen (P <0.001 after Hochberg correction for multiple comparisons). In corticosterone rats, Nos2 mRNA decreased in cortex by 68±5% and in medulla by 62±6%;Nos3 mRNA by 50±8% in cortex, and Nos1 by 29±7% in medulla (all P <0.001 after Hochberg correction). Reductions seen in kidney were supported by in situ hybridization and immunohistochemistry. Apart from a 62±2% decrease in Nos2 mRNA in adrenal of ACTH rats (corrected P <0.05), no significant changes were seen in the other nonrenal tissues for any isoform. In conclusion, we have shown for the first time that the physiological components of glucocorticoid action (ACTH and corticosterone) when given chronically in vivo reduce Nos2 and Nos3 expression in the kidney. Such changes are consistent with a role in hypertension for ACTH and corticosterone.

Expression and regulation of peroxiredoxin 5 in human osteoarthritis.

Reactive oxygen species (ROS) are implicated in the pathogenesis of osteoarthritis (OA). However, little is known about the antioxidant defence system in articular cartilage. We investigated the expression and regulation of peroxiredoxin 5 (PRDX5), a newly discovered thioredoxin peroxidase, in human normal and osteoarthritic cartilage. Our results show that human cartilage constitutively expresses PRDX5. Moreover, the expression is up‐regulated in OA. Inflammatory cytokines tumour necrosis factor α and interleukin 1 β contribute to this up‐regulation by increasing intracellular ROS production. The present study suggests that PRDX5 may play a protective role against oxidative stress in human cartilage.

Temporal expression of nitric oxide synthase isoforms in healing Achilles tendon.

We investigated the temporal expressions of the three nitric oxide synthase (NOS) isoforms by semi‐quantitative polymerase chain reaction (PCR) assays and by immunoblot analysis, following Achilles tendon transection in rats. Four days after injury, there were increases in the steady‐state levels of mRNA for all three NOS isoforms, with peaks for the inducible isoform (iNOS) (23‐fold increase) at day 4, the endothelial isoform (eNOS) (24‐fold increase) at day 7 and the neuronal isoform (bNOS) (seven‐fold increase) at day 21. The temporal expression of NOS isoforms at a protein level was consistent with the results at the mRNA level. We have previously shown a five‐fold increase in the NOS activity, as detected by 3H‐arginine to 3H‐citrulline conversion, at day 7 postinjury. These findings indicate that all three NOS isoforms are expressed during tendon healing with differential expression patterns during the various phases of tendon healing. These findings may prove clinically relevant with respect to strategies for regulating tendon healing.

The cell specific temporal expression of nitric oxide synthase isoforms during achilles tendon healing.

Abstract.Objective and design: We have previously shown that nitric oxide synthase (NOS) activity is upregulated following tendon injury, and that this activity is important to Achilles tendon healing. The aim of this study was to identify the cellular distribution of nitric oxide synthase isoforms during tendon healing.¶Material or subjects: Surgical division of the right Achilles tendon was performed in eighty-five male Sprague-Dawley rats. Healing Achilles tendons were harvested at 4, 7, 14 and 21 days following the surgery. The un-injured left Achilles tendons were used as controls. Using RNase protection assays, in situ hybridization and immunohistochemistry, mRNA and protein of NOS isoforms were evaluated.¶Results: Minimal NOS expression was found in un-injured tendon. A cell specific temporal pattern for the mRNA and protein for all three NOS isoforms was found following injury to the Achilles tendon. iNOS was maximal on day 4 in macrophages and fibroblasts. eNOS was maximal on day 4 in endothelial cells and fibroblasts. bNOS expression gradually increased up to day 21 and was found only in fibroblasts.¶Conclusions: These results suggest that all three nitric oxide synthase isoforms are expressed by fibroblasts in a coordinated temporal sequence during tendon healing. The sequential pattern of NOS expression in healing fibroblasts suggests that each NOS isoform may play a different role in the healing process and provides opportunities to modify tendon healing in the clinical setting.

Addition of nitric oxide via nitroflurbiprofen enhances the material properties of early healing of young rat Achilles tendons

Abstract:Objective and design: To determine if the addition of nitric oxide (NO) via nitroflurbiprofen (NO-flurbiprofen) would enhance rat Achilles tendon healing. Materials and methods: Sixty-five male Sprague-Dawley rats were randomly divided into NO-flurbiprofen, flurbiprofen and vehicle groups, given drugs or vehicle subcutaneously, and their right Achilles tendon divided. Histological assessment was carried out at day 5, 10, and 15 post-operation. Healing tendon biomechanical properties and hydroxyproline content were measured at day 10. Results: The healing Achilles tendon from the NO-flurbiprofen and flurbiprofen groups showed a better organization of extracellular collagenous matrix than that from the vehicle group. Flurbiprofen and NO-flurbiprofen decreased healing tendon cross-sectional area by 30% and 20%. This reduction was accompanied by a decreased failure load in the flurbiprofen group, but not the NO-flurbiprofen group. NO-flubiprofen prevented the reduction of body weight gain observed in the flubiprofen group. Conclusion: Both flurbiprofen and NO-flurbiprofen promoted better collagen reorganization during tendon healing. NO-flurbiprofen further improved tendon healing by increasing tendon stress and reducing the side effects (body weight loss) of flurbiprofen. The enhanced tendon healing by NO-flurbiprofen is likely due to the release of NO from the compound.