Subchondral bone remodeling is related to clinical improvement after joint distraction in the treatment of ankle osteoarthritis.

OBJECTIVE: In osteoarthritis (OA), subchondral bone changes alter the joint's mechanical environment and potentially influence progression of cartilage degeneration. Joint distraction as a treatment for OA has been shown to provide pain relief and functional improvement through mechanisms that are not well understood. This study evaluated whether subchondral bone remodeling was associated with clinical improvement in OA patients treated with joint distraction. METHOD: Twenty-six patients with advanced post-traumatic ankle OA were treated with joint distraction for 3 months using an Ilizarov frame in a referral center. Primary outcome measure was bone density change analyzed on computed tomography (CT) scans. Longitudinal, manually segmented CT datasets for a given patient were brought into a common spatial alignment. Changes in bone density (Hounsfield Units (HU), relative to baseline) were calculated at the weight-bearing region, extending subchondrally to a depth of 8mm. Clinical outcome was assessed using the ankle OA scale. RESULTS: Baseline scans demonstrated subchondral sclerosis with local cysts. At 1 and 2 years of follow-up, an overall decrease in bone density (-23% and -21%, respectively) was observed. Interestingly, density in originally low-density (cystic) areas increased. Joint distraction resulted in a decrease in pain (from 60 to 35, scale of 100) and functional deficit (from 67 to 36). Improvements in clinical outcomes were best correlated with disappearance of low-density (cystic) areas (r=0.69). CONCLUSIONS: Treatment of advanced post-traumatic ankle OA with 3 months of joint distraction resulted in bone density normalization that was associated with clinical improvement.

Effects of glucose on sorbitol pathway activation, cellular redox, and metabolism of myo-inositol, phosphoinositide, and diacylglycerol in cultured human retinal pigment epithelial cells.

Sorbitol (aldose reductase) pathway flux in diabetes perturbs intracellular metabolism by two putative mechanisms: reciprocal osmoregulatory depletion of other organic osmolytes e.g., myo-inositol, and alterations in NADPH/NADP+ and/or NADH/NAD+. The "osmolyte" and "redox" hypotheses predict secondary elevations in CDP-diglyceride, the rate-limiting precursor for phosphatidylinositol synthesis, but through different mechanisms: the "osmolyte" hypothesis via depletion of intracellular myo-inositol (the cosubstrate for phosphatidylinositol-synthase) and the "redox" hypothesis through enhanced de novo synthesis from triose phosphates. The osmolyte hypothesis predicts diminished phosphoinositide-derived arachidonyl-diacylglycerol, while the redox hypothesis predicts increased total diacylglycerol and phosphatidic acid. In high aldose reductase expressing retinal pigment epithelial cells, glucose-induced, aldose reductase inhibitor-sensitive CDP-diglyceride accumulation and inhibition of 32P-incorporation into phosphatidylinositol paralleled myo-inositol depletion (but not cytoplasmic redox, that was unaffected by glucose) and depletion of arachidonyl-diacylglycerol. 3 mM pyruvate added to the culture medium left cellular redox unaltered, but stimulated Na(+)-dependent myo-inositol uptake, accumulation, and incorporation into phosphatidylinositol. These results favor myo-inositol depletion rather than altered redox as the primary cause of glucose-induced aldose reductase-related defects in phospholipid metabolism in cultured retinal pigment epithelial cells.

The linked roles of nitric oxide, aldose reductase and, (Na+,K+)-ATPase in the slowing of nerve conduction in the streptozotocin diabetic rat.

Metabolic and vascular factors have been invoked in the pathogenesis of diabetic neuropathy but their interrelationships are poorly understood. Both aldose reductase inhibitors and vasodilators improve nerve conduction velocity, blood flow, and (Na+,K+)-ATPase activity in the streptozotocin diabetic rat, implying a metabolic-vascular interaction. NADPH is an obligate cofactor for both aldose reductase and nitric oxide synthase such that activation of aldose reductase by hyperglycemia could limit nitric oxide synthesis by cofactor competition, producing vasoconstriction, ischemia, and slowing of nerve conduction. In accordance with this construct, N-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase reversed the increased nerve conduction velocity afforded by aldose reductase inhibitor treatment in the acutely diabetic rat without affecting the attendant correction of nerve sorbitol and myo-inositol. With prolonged administration, N-nitro-L-arginine methyl ester fully reproduced the nerve conduction slowing and (Na+,K+)-ATPase impairment characteristic of diabetes. Thus the aldose reductase-inhibitor-sensitive component of conduction slowing and the reduced (Na+,K+)-ATPase activity in the diabetic rat may reflect in part impaired nitric oxide activity, thus comprising a dual metabolic-ischemic pathogenesis.

Modulation of basal nitric oxide-dependent cyclic-GMP production by ambient glucose, myo-inositol, and protein kinase C in SH-SY5Y human neuroblastoma cells.

Defective tissue perfusion and nitric oxide production and altered myo-inositol metabolism and protein kinase C activation have been invoked in the pathogenesis of diabetic complications including neuropathy. The precise cellular compartmentalization and mechanistic interrelationships of these abnormalities remain obscure, and nitric oxide possesses both neurotransmitter and vasodilator activity. Therefore the effects of ambient glucose and myo-inositol on nitric oxide-dependent cGMP production and protein kinase C activity were studied in SH-SY5Y human neuroblastoma cells, a cell culture model for peripheral cholinergic neurons. D-Glucose lowered cellular myo-inositol content, phosphatidylinositol synthesis, and phosphorylation of an endogenous protein kinase C substrate, and specifically reduced nitric oxide-dependent cGMP production a time- and dose-dependent manner with an apparent IC50 of approximately 30 mM. The near maximal decrease in cGMP induced by 50 mM D-glucose was corrected by the addition of protein kinase C agonists or 500 microM myo-inositol to the culture medium, and was reproduced by protein kinase C inhibition or downregulation, or by myo-inositol deficient medium. Sodium nitroprusside increased cGMP in a dose-dependent fashion, with low concentrations (1 microM) counteracting the effects of 50 mM D-glucose or protein kinase C inhibition. The demonstration that elevated D-glucose diminishes basal nitric oxide-dependent cGMP production by myo-inositol depletion and protein kinase C inhibition in peripheral cholinergic neurons provides a potential metabolic basis for impaired nitric oxide production, nerve blood flow, and nerve impulse conduction in diabetes.

Phorbol ester-mediated association of protein kinase C to the nuclear fraction in NIH 3T3 cells.

Treatment of intact NIH 3T3 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid redistribution (stabilization) of protein kinase C to the particulate fraction. Part of the enzyme activity stabilized to the membrane fraction in response to TPA can be recovered associated with nuclear-cytoskeletal components. An apparently pure nuclear fraction prepared from NIH 3T3 cells was found to contain 25-30% of the total membrane-associated protein kinase C activity when isolated in the presence of Ca2+. In untreated control cells, most of this activity found with the nuclear fraction can be extracted by chelators. Phorbol ester (TPA) treatment of NIH 3T3 cells induces the tight association of protein kinase C to the nucleus; this tightly bound activity is not dissociable by chelators and can be recovered only by solubilization with detergent. Nuclei purified from untreated human promyelocytic leukemic HL-60 cells contain higher amounts of chelator-stable, detergent-extractable protein kinase C activity compared with control NIH 3T3 cells. However, TPA treatment of HL-60 cells does not enhance the amount of protein kinase C found tightly associated with the nuclear fraction. Immunohistochemical studies with polyclonal antibodies directed against protein kinase C further indicate that TPA treatment of NIH 3T3 cells does significantly enhance the amount of protein kinase C found tightly associated with the nucleus and cytoskeleton, whereas exposure of HL-60 cells to TPA does not appreciably alter the amount of protein kinase C observed to be associated with the nuclear fraction. The TPA-mediated association (activation) of protein kinase C to the nuclear and cytoskeletal fractions with NIH 3T3 cells is further supported by the enhanced phosphorylation of specific endogenous proteins noted when purified nuclei and cytoskeletal preparations are incubated with [gamma-32P]ATP. These results suggest that tumor promoters may induce association (activation) of protein kinase C with different subcellular components to alter the availability of endogenous substrates. This may result in differential responses by different cell types during exposure to tumor promoters.

Location of the aromatic binding site and preparation of an aromatic derivative of glycogen phosphorylase.

Rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) was inactivated by 1,5-difluoro-2,4-dinitrobenzene at pH 7.6 at a rate much faster than by 1-fluoro-2,4-dinitrobenzene. The reaction was very specific at low concentration of 1,5-difluoro-2,4-dinitrobenzene. Glucose 1-phosphate, glucose 6-phosphate, AMP and ATP afforded some protection against inactivation by 1,5-difluoro-2,4-dinitrobenzene. These results and kinetic analyses of the modified enzyme were used to locate the binding site for aromatic compounds in phosphorylase. The above ligands and aromatic compounds are shown to bind on the enzyme in the same region which is located near the monomer/monomer interface. An apparently homogeneous dinitrodiphenyzene derivative of phosphorylase b with only one group per dimeric enzyme and having 50% of the catalytic activity was prepared. This derivative in which the subunits were not cross-linked by the reagent was devoid of the homotropic cooperativity for the substrate or activator sites even in the presence of allosteric inhibitors. Glucose behaved quite differently from other ligands in its effect on modification and on the kinetics of the modified enzyme. The significance of the glucose site is discussed.

Dioctanoylglycerol regulation of cytosolic Ca2+ by protein kinase C-independent mechanism in HIT T-15 islet cells.

The effect of activators of protein kinase C (PKC) on cytosolic concentration of free Ca2+ [( Ca2+]i) was assessed in insulin-secreting islet cell line HIT T-15. Dioctanoylglycerol (DiC8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) evoked activation of PKC. Basal [Ca2+]i was 65-160 nM. DiC8 induced triphasic increases in [Ca2+]i; phase 2 was the most prominent and consistent one. With 25-150 microM DiC8, [Ca2+]i increased in a dose-dependent manner during phase 2; half-maximal stimulatory dose was 53 microM. TPA did not evoke any increase in [Ca2+]i. Staurosporine, sphingosine, and H7, which are inhibitors of PKC, did not block DiC8-induced rise in [Ca2+]i. DiC8-induced rise in [Ca2+]i was also seen in cells that had been depleted of PKC by prior exposure to TPA. DiC8-induced rise in [Ca2+]i still occurred in the presence of the Ca(2+)-channel blocker verapamil or when the extracellular Ca2+ had been reduced from 2.5 mM to 30 nM by EGTA. Three immediate metabolites of DiC8, monooctanoylglycerol, octanoate, and glycerol, did not evoke any change in [Ca2+]i. Monooleoylglycerol and R59022, which induce increases in endogenous diacylglycerol (DAG) by inhibiting DAG kinase, evoked increases in [Ca2+]i. DiC8 did not cause any change in inositol 1,4,5-trisphosphate levels. DiC8 evoked biphasic increases in insulin release; the second-phase increase in [Ca2+]i preceded the late phase of insulin secretion. Exogenous DAGs should be used with caution in assessing PKC function. Changes in the generation in DAGs must be included among the mechanisms by which Ca2+ homeostasis is regulated in islet cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin release in RINm5F cells and glyceraldehyde activation of protein kinase C.

In the insulin-secreting, glucose-insensitive islet cell subclone RINm5F, the distribution of protein kinase C (PKC) activity in the cytosol and membrane fractions was determined, and the activation of the enzyme, as reflected in its translocation to the membrane fraction, was characterized in conjunction with insulin release. DL-Glyceraldehyde (15 mM) evoked a rapid redistribution of PKC from the cytosol to the membrane fraction; insulin release increased concomitantly. When monitored over 5 min with 15 mM glyceraldehyde, membrane stabilization of PKC reached a maximum at 30 s and decreased thereafter; insulin release occurred at a high rate for the first 15 s and diminished thereafter. With 2-20 mM glyceraldehyde, a dose-dependent increase in membrane stabilization of PKC occurred and was accompanied by a matching increase in insulin release. Exogenous 1,2-dioctanoyl-sn-glycerol (100 microM) induced a rapid membrane stabilization of PKC and concomitant stimulation of insulin release. Glucose (15 mM) failed to evoke any redistribution of PKC or release of insulin. Depletion of total PKC activity by 95% induced by 18-h incubations with 2 microM 12-O-tetradecanoylphorbol-13-acetate resulted in a 67-91% reduction in glyceraldehyde-induced insulin release. We conclude that in the RINm5F islet beta-cell subclone 1) the rapid activation of PKC, which occurs in response to the administration of glyceraldehyde, a nutrient secretagogue, plays an amplifying role in the initiation of stimulated insulin release; and 2) the failure of the activation of PKC may be responsible for the insensitivity to glucose.

Diminished specific activity of cytosolic protein kinase C in sciatic nerve of streptozocin-induced diabetic rats and its correction by dietary myo-inositol.

The impaired Na(+)-K(+)-ATPase activity in peripheral nerve from diabetic rats is prevented by dietary myo-inositol (MI) supplementation in vivo and corrected by protein kinase C (PKC) agonists in vitro, suggesting that PKC may mediate the effects of nerve MI depletion on Na(+)-K(+)-ATPase activity. However, little is known about the effect of diabetes on PKC activity or peptide in rat peripheral nerve. Therefore, the effect of streptozocin-induced diabetes and dietary MI supplementation on the activity and distribution of PKC in rat sciatic nerve homogenates and cytosolic and particulate fractions was explored with histone phosphorylation assay and Western-blot analysis. PKC activity but not peptide was selectively decreased in the cytosolic fraction by streptozocin-induced diabetes, and this abnormality was partially corrected by dietary MI supplementation. These results suggest that altered MI metabolism may affect nerve PKC specific activity, and this alteration may play a role in reduced Na(+)-K(+)-ATPase activity and blunted regenerative response in diabetic nerve.

Diacylglycerol inhibits potassium-induced calcium influx and insulin release by a protein kinase-C-independent mechanism in HIT T-15 islet cells.

We reported previously that in pancreatic islet cells, certain diacylglycerols (DGs) evoke increases in cytosolic calcium ([Ca2+]i), mainly by intracellular mobilization. We now examined the effects of DGs on the increase in [Ca2+]i due to Ca2+ influx. In the insulin-secreting HIT T-15 islet cell line, cell membrane depolarization using 40 mM KCl evoked a 2- to 3-fold increase in [Ca2+]i, which lasted several minutes. A cell-permeable DG, 1,2-dioctanoylglycerol (DiC8; 10 microM) induced a 12 +/- 4% rise in [Ca2+]i, which did not occur in the absence of extracellular Ca2+ or in the presence of verapamil; this effect was not protein kinase-C (PKC) dependent, because it was not altered by the addition of the PKC inhibitor staurosporine or by using PKC-depleted cells. When DiC8 was added first, the KCl-induced increase in [Ca2+]i was inhibited in a dose-dependent manner (100% at 10-15 microM DiC8); this effect was PKC independent. At a concentration of 10 microM, other synthetic DGs, 1,2-dihexanoylglycerol (DiC6), 1,2-didecanoylglycerol (DiC10), or 1-oleoyl-2-acetylglycerol, inhibited the KCl-induced rise in [Ca2+]i to 15 +/- 4%, 47 +/- 7%, and 51 +/- 5% of the control value, respectively. R59022 (10 microM), which inhibits DG kinase and causes accumulation of endogenous DGs, inhibited the KCl-induced rise in [Ca2+]i to 2 +/- 0.2% of the control value; this inhibition was not affected by staurosporine. In anchored cells, KCl stimulated insulin release (959 +/- 88 microU/mg protein above the control value); 20 microM DiC6 or DiC8 attenuated KCl-induced insulin release by 68% and 31% of the control value, respectively; DiC10 or 1-oleoyl-2-acetylglycerol had no effect. R59022 inhibited KCl-induced insulin release by 90% of the control value. We conclude that in HIT T-15 cells, DGs may serve as positive and negative modulators of [Ca2+]i, apparently by complex and PKC-independent mechanisms. These divergent actions of DGs on islet cell Ca2+ balance together with the accompanying activation of PKC affect insulin release in a complex manner.

2-Chloroadenosine reverses hyperglycemia-induced inhibition of phosphoinositide synthesis in cultured human retinal pigment epithelial cells and prevents reduced nerve conduction velocity in diabetic rats.

The effect of the adenosine (AD) analog 2-chloroadenosine (C-AD) on glucose-induced inhibition of phosphoinositide synthesis was studied in human retinal pigment epithelial (RPE) cells by monitoring the level of the phosphatidylinositol (PI) synthase substrate, cytidine diphosphate diglyceride (CDP-DG). In high-aldose reductase (AR)-expressing RPE 91 cells, C-AD decreased CDP-DG at 5 mmol/L glucose and reversed the increase by 20 mmol/L glucose. AD deaminase (ADA), which inactivates endogenously released AD, potentiated the hyperglycemia-induced increase in CDP-DG. Theophylline, an AD-A1 and AD-A2 receptor antagonist, caused an increase in CDP-DG at 20 mmol/L glucose. C-AD did not alter CDP-DG in low-AR-expressing RPE 45 cells, but did decrease CDP-DG after cells were conditioned in 300 mmol/L glucose for 1 week (which induces AR). The mechanism by which AD regulates PI synthase in cells with high AR activity is unknown, but it is independent of Gi or Gs proteins, adenylate cyclase and phospholipase C (PLC) activation, myo-inositol (MI) uptake, or MI efflux. Administration of C-AD to streptozotocin-induced diabetic rats prevented the slowing of motor nerve conduction velocity (MNCV). Thus, AD derivatives, which reverse a glucose-induced deficit in phosphoinositide metabolism, might serve as a useful pharmacological tool to intervene in hyperglycemia-induced diabetic complications.

Probucol improves antioxidant activity and modulates development of diabetic cardiomyopathy.

To examine the role of free radicals in diabetic cardiomyopathy, myocardial antioxidants as well as lipid peroxide content were examined in rats made diabetic with a single injection of streptozotocin (65 mg/kg i.v). At 4 wk, the left ventricular peak systolic (LVSP) as well as aortic pressures were depressed in the diabetic group. Hearts from diabetic animals showed about a 100% increase in thiobarbituric acid reactive substances (TBARS), indicating increased lipid peroxidation. This was accompanied by about a 50% decrease in superoxide dismutase (SOD) and 60% decrease in glutathione peroxidase (GSHPx) enzyme activities. Catalase activity in these hearts showed a small but significant increase. Treatment with probucol (10 mg/kg i.p., on alternate days), a known lipid-lowering drug with strong antioxidant properties, was initiated 1 d after the induction of diabetes and was continued for 4 wk. In probucol-treated diabetic animals, LVSP was not different from controls. Probucol treatment caused a small but significant improvement in serum insulin and decrease in glucose levels as well as increased myocardial SOD, GSHPx, and catalase activities with a concomitant decrease in TBARS in the diabetic animals. These data provide evidence that diabetic cardiomyopathy is associated with an antioxidant deficit, and a better cardiac function due to treatment with probucol may be related to the improved insulin levels as well as maintenance of the antioxidant status of the heart.

Antioxidant protection against adrenaline-induced arrhythmias in rats with chronic heart hypertrophy.

Effects of vitamin E on adrenaline-induced arrhythmias were examined in rats with chronic heart hypertrophy subsequent to narrowing of the abdominal aorta. After 60 weeks of pressure overload, the rats showed an increase of about 21% in heart/body weight ratio and a small but significant rise in left ventricular end diastolic pressure (LVEDP) (sham control 1.7 +/- 0.67 mmHg; hypertrophy 7.1 +/- 2.7 mmHg) without any change in left ventricular peak systolic pressure (LVSP). Intravenous infusion of adrenaline caused rhythm disorders in a dose-dependent manner and pathological arrhythmias (occurrence of six premature ventricular complexes/min) were observed at doses of 2.9 +/- 0.6 and 3.8 +/- 1.0 micrograms/kg of the drug in control and hypertrophy animals, respectively. Administration of two doses of vitamin E (50 mg/kg intraperitoneally), given 24 h and 1 h before adrenaline infusion, significantly increased the amount of adrenaline required to produce pathological arrhythmias (control 8.0 +/- 3.0; hypertrophy 7.7 +/- 2.0 micrograms/kg). Vitamin E pretreatment did not have any detrimental effect on the pressure readings nor did it have any influence on adrenaline-induced pressure changes. The data suggest that a combination therapy with vitamin E may allow therapeutic use of higher concentrations of adrenaline required to improve function in failing hearts with a reduced risk of arrhythmias.

Acute articular fracture severity and chronic cartilage stress challenge as quantitative risk factors for post-traumatic osteoarthritis: illustrative cases.

Novel biomechanical methods have been developed to objectively measure acute fracture severity (from inter-fragmentary surface area) and chronic contact stress challenge (from patient-specific finite element analysis) in articular fractures. These new methods help clarify the pathomechanics of the development of post-traumatic osteoarthritis, and can contribute directly to the clinical care of patients. In this manuscript, the value of these two new measures is demonstrated in three illustrative tibial plafond fracture cases, in which both metrics are correlated with cartilage status and with patient outcomes at a minimum of two years after injury. These clinical cases demonstrate the utility of new biomechanical variables to advance clinical research and patient care, by providing a basis to predict outcome and select treatment.

The complications of systemic corticosteroid therapy in the elderly. A retrospective study.

The complications of long-term corticosteroid therapy were reviewed in 100 elderly patients who were treated for chronic obstructive airways disease (n = 76), rheumatoid arthritis (n = 19) and ulcerative colitis (n = 5). The incidence of side effects was high (40%) and appeared to be dose-related. Osteoporosis (16%) and hypertension (12%) were the most common. Hypokalaemia occurred infrequently despite the fact that 69 patients were also prescribed diuretics. A further group of 36 patients receiving corticosteroids for polymyalgia rheumatica and giant cell arteritis also seemed to demonstrate a dose-related effect on the incidence of complications although this could not be confirmed statistically.

Night sweats--presentation of an often forgotten diagnosis.

Night sweats are not uncommon complaints in the elderly and they are readily associated with disorders of catecholamine excess, solid malignancies and tuberculosis. We report night sweats as the presenting feature of giant cell arteritis.