Renal artery revascularization in patients with atherosclerotic renal artery stenosis and impaired renal function: conservative management versus renal artery stenting.

BACKGROUND: The impact of percutaneous renal artery angioplasty and stenting (PTRAS) for treatment of atherosclerotic renal artery stenosis (ARAS) is not fully understood, especially in patients with chronic kidney disease (CKD). The goal of this study was to compare renal outcomes in patients treated with PTRAS with those managed conservatively. METHODS: Retrospective cohort study of 118 prevalent patients with significant ARAS and moderate-to-severe CKD who were treated medically (n = 71) or with PTRAS (n = 47). The primary endpoint was change in renal function over the first year after diagnosis/treatment. RESULTS: Average age was 73 +/- 8 years, baseline glomerular filtration rate was 37 +/- 15 ml/min/1.73 m2, and average follow-up was 34 months. Baseline characteristics were similar between the two groups, with the exception of higher diastolic blood pressure in the PTRAS group (75 versus 70 mmHg, p = 0.028). There were no significant differences between the two groups during follow-up. The decline in glomerular filtration rate was similar in both groups (-1.6 ml/min/1.73 m2 in the medical group versus -1.4 ml/min/1.73 m2 in the PTRAS group, p = 0.938). Multivariate models did not indicate an association between treatment modality and changes in renal function or rates of ESRD or death. CONCLUSION: In patients with advanced kidney disease, medical therapy and renal artery stenting appear comparable in stabilizing renal function for ARAS.

A combination of a chemically modified doxycycline and a bisphosphonate synergistically inhibits endotoxin-induced periodontal breakdown in rats.

BACKGROUND: Chemically modified non-antimicrobial tetracyclines (CMTs) have been shown to inhibit pathologically elevated collagenase (and other matrix metalloproteinase, MMP) activity and bone resorption in vivo and in vitro. METHODS: In the current study, suboptimal doses of CMT-8 (a non-antimicrobial chemically modified doxycycline) and a bisphosphonate (clodronate, an anti-bone resorption compound) were administered daily, either as a single agent or as a combination therapy, to rats with experimental periodontitis induced by repeated injection of bacterial endotoxin (LPS) into the gingiva. At the end of the 1-week protocol, the gingival tissues were dissected, extracted, and the extracts analyzed for MMPs (collagenases and gelatinases) and for elastase, and the defleshed jaws were morphometrically analyzed for alveolar bone loss. RESULTS: LPS injection significantly (P<0.001) increased alveolar bone loss and increased collagenase (MMP-8), gelatinase (MMP-9), and elastase activities. Treatment of the LPS-injected rats with suboptimal CMT-8 alone or suboptimal clodronate alone produced slight reductions in the tissue-destructive proteinases and no significant reductions in alveolar bone loss. However, a combination of suboptimal CMT-8 and clodronate "normalized" the pathologically elevated levels of MMPs, elastase, and alveolar bone loss, indicating synergistic inhibition of tissue breakdown in this animal model of periodontitis. CONCLUSIONS: Combination of a CMT and a bisphosphonate may be a useful treatment to optimally suppress periodontal destruction and tooth loss and in other tissue-destructive inflammatory diseases such as arthritis.

The lipophilicity, pharmacokinetics, and cellular uptake of different chemically-modified tetracyclines (CMTs).

CMTs are analogs of tetracyclines, which are chemically modified to eliminate their antimicrobial efficacy but which retain their inhibitory activity against matrix metalloproteinases. These compounds have been found to inhibit connective tissue breakdown in animal models of diseases such as periodontitis, arthritis and cancer. Because CMTs exhibit different in vivo efficacy in these various models of disease, the current study compared their pharmacokinetics and other properties as follows: Adult male Sprague-Dawley rats were administered by oral gavage a single dose of 5mg of different CMTs suspended in 1 ml 2% carboxymethyl-cellulose, and blood samples were collected from 1-48 hours after dosing. The sera were extracted, then analyzed by HPLC using a C-18 reverse-phase column. The results showed that the peak concentrations (C(max)) in rat sera 1-12 hours after oral administration of CMTs -1, -2,-3, -4,-5,-6,-7,-8 and doxycycline were 5.5, 0.7, 4.6, 6.2, 0.8, 0.7, 9.0 (note: the 3 peaks detected for CMT-7 were combined), 15.0 and 0.9 microg/ml, respectively. Their in vivo half-lives (t(1/2)) were 11, 5, 22, 11, 32, 15, 37, 38, and 17 hours, respectively. Of the anticollagenase CMTs tested, CMT-8 showed the greatest C(max) and t(1/2)values, followed by CMTs-3, -1, -4, and perhaps -7; CMTs-2, -5, and -6 exhibited much lower levels in serum. The relative lipophilicities of the 8 CMTs and doxycycline were tested by examining their extractability in octanol. The results showed that CMT-2, -5, and -6 had the lowest partition coefficients using this organic solvent, while CMT-3 was the most lipophilic. The lipophilicity of the different CMTs was also positively correlated (r(2)=0.767, P<0.05) to peak serum concentrations (C(max)), but not to their serum half-lives (r(2)=0.25,P=0.49). This property of the different CMTs was also found to be positively correlated to their ability to enter into human whole blood cells in vitro (r2=0.95, P<0.001). Since CMT-8, as well as CMTs-3 and -1, consistently exhibited the greatest in vivo efficacy in animal models of tissue breakdown, this may reflect, at least in part, their favorable pharmacokinetics and tissue uptake.

Functional sites of chemically modified tetracyclines: inhibition of the oxidative activation of human neutrophil and chicken osteoclast pro-matrix metalloproteinases.

OBJECTIVE: We studied the relative ability of 6 different chemically modified non-antimicrobial analogs of tetracycline (CMT) to inhibit human and chicken matrix metalloproteinases (MMP) in vitro. The ability of tetracycline and its analogs to inhibit MMP appears to depend on the Ca++/Zn++ binding site at C11 (carbonyl oxygen) and C12 (OH group) of the molecule, which is lacking in CMT-5, the pyrazole derivative of tetracycline. This significant property of CMT-5 was used to differentiate between the effects of CMT on already active MMP versus the oxidative activation of latent MMP (pro-MMP). METHODS: Cultured chicken osteoclast conditioned medium and purified human neutrophil progelatinase (MMP-9) and pro-collagenase (MMP-8) were assayed for proteinase activities using gelatin and collagen, respectively. The pro-MMP were activated either by preincubation with 1 mM aminophenylmercuric acetate (APMA) or 100 microM sodium hypochlorite (NaOCI). CMT were added either to the preincubation mixtures together with NaOCl or after activation of pro-MMP with NaOCl. RESULTS: All CMT tested, except CMT-5, inhibited APMA or NaOCl activated pro-MMP. However, CMT-5 (like the other CMT), inhibited the oxidative activation of pro-MMP by NaOCl when added together by scavenging the reactive oxygen species. The degradation of type-I collagen by chicken osteoclast conditioned medium was probably due to MMP-2 and/or MMP-13. CONCLUSION: Oxidative activation of pro-MMP may be crucial during soft tissue/bone destruction in the inflammatory diseases, including the arthritides. Our results indicate that the Ca++/Zn++ binding site of CMT is not essential for inhibition of the oxidative activation of pro-MMP.

Effects of tetracyclines on bone metabolism.

The anti-resorptive properties of tetracyclines (TCs) and their non-antimicrobial, chemically modified analogues (CMTs) have enormous therapeutic potential in medicine and dentistry. Osseous destructive diseases associated with excessive mammalian collagenase (matrix metalloproteinase) activity and collagen breakdown include malignancy, arthritis, and periodontitis. However, apart from the significant antimatrix metalloproteinase effects of TCs, TCs/CMTs are also potent inhibitors of osteoclast function (i.e., anti-resorptive). Thus, TCs can affect several parameters of osteoclast function and consequently inhibit bone resorption by (1) altering intracellular calcium concentration and interacting with the putative calcium receptor; (2) decreasing ruffled border area; (3) diminishing acid production; (4) diminishing the secretion of lysosomal cysteine proteinases (cathepsins); (5) inducing cell retraction by affecting podosomes; (6) inhibiting osteoclast gelatinase activity; (7) selectively inhibiting osteoclast ontogeny or development; and (8) inducing apoptosis or programmed cell death of osteoclasts. TCs/CMTs, as anti-resorptive drugs, may act similarly to bisphosphonates and primarily affect osteoclast function.

Halitosis. A common oral problem.

Halitosis is caused primarily by bacterial putrefaction and the generation of volatile sulfur compounds. Ninety percent of patients suffering from halitosis have oral causes, such as poor oral hygiene, periodontal disease, tongue coat, food impaction, unclean dentures, faulty restorations, oral carcinomas, and throat infections. The remaining 10 percent of halitosis sufferers have systemic causes that include renal or hepatic failure, carcinomas, diabetes or trimethylaminuria. Modern analytical and microbiological techniques permit diagnosis of bad breath. Management of halitosis involves maintaining proper oral hygiene, and periodontal treatment, including tongue brushing.

Regulation of extracellular calcium sensing in rat osteoclasts by femtomolar calcitonin concentrations.

Certain eukaryotic cells can sense changes in their extracellular Ca2+ concentration through molecular structures termed Ca(2+)-sensing receptors (CaRs). We have shown recently that in the bone-resorbing osteoclast, a unique cell surface-expressed ryanodine receptor (RyR), functions as the CaR. The present study demonstrates that the sensitivity of this receptor is modulated by physiological femtomolar concentrations of the bone-conserving hormone, calcitonin. Calcitonin was found to inhibit cytosolic Ca2+ responses to both Ca2+ and Ni2+. The latter inhibition was mimicked by amylin (10(-12) M), calcitonin gene-related peptide (10(-12) M), cholera toxin (5 micrograms/l) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) (2.5 x 10(-4) or 5 x 10(-4) M) and was reversed by the protein kinase A phosphorylation inhibitor, IP-20. Finally, using a quench flow module, we showed that cellular cAMP levels rise to a peak within 25 ms of calcitonin application; this is consistent with the peptide's rapid effect on CaR activation. We conclude, therefore, that cAMP plays a critical role in the control of CaR function by calcitonin.

Adaptations of intestinal nutrient transport to chronic caloric restriction in mice.

Lifelong caloric restriction increases median and maximum life span and retards the aging process in many organ systems of rodents. Because the small intestine absorbs a reduced amount of nutrients each day, does lifelong caloric restriction induce adaptations in intestinal nutrient transport? We initially compared intestinal transport of sugars and amino acids between 24-mo-old mice allowed free access to food [ad libitum (AL)] and those provided a calorically restricted [40% less than ad libitum (CR)] diet since 3 mo of age. We found that CR mice had significantly greater transport rates for D-glucose, D-fructose, and several amino acids and had significantly lower villus heights. Total intestinal absorptive capacities for D-glucose, D-fructose, and L-proline were each 40-50% greater in CR mice; absorptive capacity normalized to metabolic mass (body weight 0.75) was approximately 80% greater in CR mice. Comparison of uptakes in aged AL and CR mice with previously published results in young AL mice suggests that caloric restriction delays age-related decreases in nutrient transport. In contrast to published studies in hibernation and starvation, chronic caloric restriction enhances not only uptake per milligram but also uptake per centimeter. We then switched 24-mo-old AL mice to a calorie-restricted diet for 1 mo and found that short-term caloric restriction has no effect on intestinal nutrient transport, intestinal mass, and total absorptive capacity. Thus chronic but not short-term caloric restriction increases intestinal nutrient transport rates in aged mice, and the main mechanism underlying these increases is enhanced transport rates per unit intestinal tissue weight.

A ryanodine receptor-like molecule expressed in the osteoclast plasma membrane functions in extracellular Ca2+ sensing.

Ryanodine receptors (RyRs) reside in microsomal membranes where they gate Ca2+ release in response to changes in the cytosolic Ca2+ concentration. In the osteoclast, a divalent cation sensor, the Ca2+ receptor (CaR), located within the cell's plasma membrane, monitors changes in the extracellular Ca2+ concentration. Here we show that a RyR-like molecule is a functional component of this receptor. We have demonstrated that [3H] ryanodine specifically binds to freshly isolated rat osteoclasts. The binding was displaced by ryanodine itself, the CaR agonist Ni2+ and the RyR antagonist ruthenium red. The latter also inhibited cytosolic Ca2+ elevations induced by Ni2+. In contrast, the responses to Ni2+ were strongly potentiated by an antiserum Ab129 raised to an epitope located within the channel-forming domain of the type II RyR. The antiserum also stained the surface of intact, unfixed, trypan blue-negative osteoclasts. Serial confocal sections and immunogold scanning electron microscopy confirmed a plasma membrane localization of this staining. Antiserum Ab34 directed to a putatively intracellular RyR epitope expectedly did not stain live osteoclasts nor did it potentiate CaR activation. It did, however, stain fixed, permeabilized cells in a distinctive cytoplasmic pattern. We conclude that an RyR-like molecule resides within the osteoclast plasma membrane and plays in important role in extracellular Ca2+ sensing.

An ultrastructural evaluation of the effects of cysteine-proteinase inhibitors on osteoclastic resorptive functions.

This study was designed to evaluate the effects of specific and potent cathepsin inhibitors on osteoclastic resorptive functions in vitro by means of a novel ultrastructural assay system. Mouse bone marrow cell-derived osteoclasts were suspended on dentine slices and cultured for 48 hours in the presence of either E-64 (a generalized cysteine proteinase inhibitor) or Z-Phe-Phe-CHN2 (a selective cathepsin L inhibitor). After the removal of cultured osteoclasts, co-cultured dentine slices were examined using electron microscopy: backscattered (BSEM), scanning (SEM), and atomic force (AFM). In morphometric analyses of BSEM images, there were no significant differences in the areas of demineralized dentine surfaces between control and inhibitor-treated groups, suggesting that cathepsin inhibitors had no effect on dentine demineralization by cultured osteoclasts. However, in SEM and AFM observations, both inhibitors remarkably reduced to the same extent, the formation of deep resorption lacunae on dentine slices that had resulted from degradation of matrix collagen. In addition, Z-Phe-Phe-CHN2 treatment produced deeper, ring-like grooves with little collagen exposure in shallow resorption lacunae. These results strongly suggest that (1) cathepsins released by osteoclasts are involved in the formation of deep resorption lacunae, and (2) cathepsin L plays a key role in bone resorption.

Estrogen modulation of osteoclast lysosomal enzyme secretion.

Osteoclast-mediated bone resorption is accomplished by secretion of lysosomal proteases into an acidic extracellular compartment. We have previously demonstrated that avian osteoclasts and human osteoclast-like giant cell tumor cells respond in vitro to treatment with 17 beta-estradiol (17 beta-E2) by decreased bone resorption activity. To better understand the mechanism by which this is accomplished, we have investigated the effects of 17 beta-E2 treatment on lysosomal enzyme production and secretion by isolated avian osteoclasts and multinucleated cells from human giant cell tumors in vitro. Isolated cells were cultured with bone particles in the presence of either vehicle or steroid. The conditioned media and cells were harvested, and the levels of cathepsin B, cathepsin L, beta-glucuronidase, lysozyme, and tartrate-resistant acid phosphatase (TRAP) activities were determined. There was a steroid dose-dependent decrease in secreted levels of these enzymes. Cell-associated levels of cathepsin L, beta-glucuronidase, and lysozyme decreased; whereas cell-associated levels of cathepsin B and TRAP increased. These changes were measurable at 10(-10) M and maximal at 10(-8) M 17 beta-E2. The changes were detectable at 4-18 h of treatment and increased through 24 h of treatment. The response was steroid specific, since the inactive estrogen isomer, 17 alpha-E2, failed to alter the activity levels. Moreover, the effects of 17 beta-E2 were blocked when the cells were treated simultaneously with the estrogen antagonist ICI182-780 in conjunction with 17 beta-E2. Human osteoclast-like cells obtained from giant cell tumors of bone responded similarly to estrogen with respect to cathepsin B, cathepsin L, and TRAP activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of gelatinases/type IV collagenases in jaw cysts.

The molecular mechanisms of jaw cyst expansion probably involve interactions of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). In this study, molecular species of gelatinases present in neutral salt extracts of cyst walls and cyst fluids were characterized by functional activity measurements (type I gelatin and alpha-casein zymography) and immunologically (Western-blotting). The effects of various protein thiol-group or cysteine-switch reactants involved in the activation of collagenases were studied on cyst gelatinases and a gelatinases purified from human gingival fibroblasts (72 kD MMP-2), gingival keratinocytes (92 kD MMP-9) and polymorphonuclear neutrophilic leukocytes (92 kD MMP-9). Western-blotting revealed the presence of both 92 kD (MMP-9) and 72 kD (MMP-2) gelatinases in cyst wall extracts and cyst fluids. Western-blot studies further suggested that jaw cyst gelatinases were only in part complexed with and thus inhibited by TIMP-1 or TIMP-2, suggesting that both MMP-9 and MMP-2 may participate in cyst expansion. MMP-2 was also partially fragmented to a 68 kD form and additional lower molecular weight proteinases (< 60 kD) were detected by alpha-casein zymography and by Western-blotting, suggesting proteolytic fragmentation. MMP-9 was at least partially activated by all protein-thiol group reactants and rather resistant to oxidative inhibition by hypochlorite (NaOCl); in contrast, MMP-2 was activated by APMA but not at all by gold thioglucose (GTG) and was clearly inactivated by hypochlorite (NaOCl).(ABSTRACT TRUNCATED AT 250 WORDS)

The nonantimicrobial properties of tetracycline for the treatment of periodontal disease.

Tetracyclines have nonantimicrobial properties that appear to modulate host response. In that regard, tetracyclines and their nonantimicrobial chemically modified analogues (chemically modified tetracycline molecules [CMTs]) inhibit the extracellular activity of mammalian neutrophil and osteoblast collagenases. The activity of this matrix metalloproteinase appears crucial in the destruction of collagen. Apart from its anticollagenase effect, tetracyclines are also potent inhibitors of osteoclast function. Several recent studies have also addressed the therapeutic potential of tetracyclines and CMTs in periodontal disease. These drugs reduced excessive gingival collagenase activity and severity of periodontal breakdown in rats infected with Porphyromonas gingivalis and in diabetic rats. CMT was not associated with the emergence of resistant microorganisms. In human double-blind clinical trials, low-dose doxycycline therapy substantially reduced collagenase activity in the gingival and crevicular fluid, and prevented the loss of attachment in adult periodontitis without the emergence of doxycycline-resistant microorganisms. Tetracyclines and CMTs have enormous therapeutic potential because these drugs can inhibit the activity of matrix metalloproteinases as well as osteoclast function, and thus prevent the degradation of osseous connective tissues in periodontal as well as arthritic diseases.

Reactive oxygen species activate and tetracyclines inhibit rat osteoblast collagenase.

Recent studies have demonstrated that tetracyclines (TCs) scavenge reactive oxygen species (ROS). Hypochlorous acid (HOCl), an ROS produced by neutrophils, has been shown to activate neutrophil procollagenase. The objective of the present study was to determine whether (1) HOCl also activated osteoblast procollagenase and (2) TCs inhibited this enzyme in the presence of HOCl. HOCl (5 microM) activated the proenzyme approximately sixfold (P < 0.01) from the medium of PTH-treated UMR-106-01 osteoblastic osteosarcoma cells as determined by functional collagenase assay (3H-methyl-labeled collagen substrate). Doxycycline (50-400 microM) and chemically modified tetracycline, CMT-1 (100-400 microM), significantly inhibited collagenase activity 50-90% and 40-80%, respectively, in the presence of 5 microM HOCl. Concentrations of 6-25 microM doxycycline and 10-50 microM CMT-1 had no significant effect. Furthermore, an excess concentration of cation (50 mM CaCl2 or 50 microM ZnCl2) added to the incubation mixtures containing either doxycycline or CMT-1 did not restore collagenase activity, as demonstrated by SDS-PAGE-fluorography. These data suggested that TCs reduced available HOCl and thus prevented the hypochlorous acid conversion of the osteoblast proenzyme to active collagenase. TCs may have therapeutic potential in the treatment of periodontitis and other diseases by several mechanisms that inhibit pathologic collagen breakdown.

Effect of tetracyclines which have metalloproteinase inhibitory capacity on basal and heparin-stimulated bone resorption by chick osteoclasts.

Several tetracyclines (TETs) are potent inhibitors of collagenase (CGase) and can inhibit connective tissue degradation in a variety of inflammatory and degenerative disorders. The role of CGase in bone resorption by osteoclasts (OC) remains unclear. Disaggregated OCs from chick embryos were cultured for 24 h on devitalized bovine cortical bone +/- heparin in the presence of various TETs. Doxycycline (Dox) inhibited pit formation in a dose-dependent manner. CMT, a TET derivative which inhibits matrix metalloproteinases (MMPs) but is not antimicrobial, also inhibited chick OC bone resorption. Heparin markedly stimulated bone resorption at 5 micrograms/ml, which was reversed by Dox, 5 micrograms/ml. TETs can reversibly inhibit both basal and heparin-stimulated bone resorption by chick OCs. These findings suggest that MMPs may play a role in osteoclastic bone resorption, and that safe and effective inhibitors of MMPs, including certain TETs, might have a potential therapeutic role.

Blocking periodontal disease progression by inhibiting tissue-destructive enzymes: a potential therapeutic role for tetracyclines and their chemically-modified analogs.

Tetracyclines (TCs) have wide therapeutic usage as antimicrobial agents; these drugs (e.g., minocycline, doxycycline) remain useful as adjuncts in periodontal therapy. However, TCs also have non-antimicrobial properties which appear to modulate host response. In that regard, TCs and their chemically-modified analogs (CMTs) have been shown to inhibit the activity of the matrix metalloproteinase (MMP), collagenase. The activity of this enzyme appears crucial in the destruction of the major structural protein of connective tissues, collagen. Such pathologic collagenolysis may be a common denominator in tissue destructive diseases such as rheumatoid and osteoarthritis, diabetes mellitus, bullous dermatologic diseases, corneal ulcers, and periodontitis. The mechanisms by which TCs affect and, possibly, diminish bone resorption (a key event in the pathogenesis of periodontal and other diseases) are not yet understood. However, a number of possibilities remain open for investigation including the following: TCs may 1) directly inhibit the activity of extracellular collagenase and other MMPs such as gelatinase; 2) prevent the activation of its proenzyme by scavenging reactive oxygen species generated by other cell types (e.g. PMNs, osteoclasts); 3) inhibit the secretion of other collagenolytic enzymes (i.e. lysosomal cathepsins); and 4) directly affect other aspects of osteoclast structure and function. Several recent studies have also addressed the therapeutic potential of TCs and CMTs in periodontal disease. These drugs reduced excessive gingival collagenase activity and severity of periodontal breakdown in rats infected with Porphyromonas gingivalis and in diabetic rats. Furthermore, the latter drug (CMT) was not associated with the emergence of TC-resistant microorganisms.(ABSTRACT TRUNCATED AT 250 WORDS)