Biomedical subjects

S D Shapiro

Publications and source records attributed to S D Shapiro.

At least 73 records · Page 4Linked to original sources

PubMed1998

Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis.

Exposure to the chemotherapeutic drug bleomycin leads to pulmonary fibrosis in humans and has been widely used in animal models of the disease. Using C57BL/6 bleomycin-sensitive mice, pulmonary fibrosis was induced by multiple intraperitoneal injections of the drug. An increase in the relative amounts of steady-state alpha1(I) procollagen, alpha1(III) procollagen, and fibronectin mRNA as well as histopathological evidence of fibrosis was observed. The effect of bleomycin on the expression of the enzymes responsible for extracellular matrix degradation, the matrix metalloproteinases (MMPs), and their inhibitors (TIMPs), was selective and showed temporal differences during the development of fibrosis. Of the MMPs tested, bleomycin treatment resulted in the up-regulation of gelatinase A and macrophage metalloelastase gene expression in whole-lung homogenates, whereas gelatinase B, stromelysin-1, and interstitial collagenase gene expression was not significantly changed. Timp2 and Timp3, the murine homologues of the respective TIMP genes, were constitutively expressed, whereas Timp1 was markedly up-regulated during fibrosis. The strong correlation between enhanced extracellular matrix gene expression, differential MMP and TIMP gene expression, and histopathological evidence of fibrosis suggest that dysregulated matrix remodeling is likely to contribute to the pathology of bleomycin-induced pulmonary fibrosis.

Animals↗

PubMed1997

Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice.

To determine which proteinases are responsible for the lung destruction characteristic of pulmonary emphysema, macrophage elastase-deficient (MME-/-) mice were subjected to cigarette smoke. In contrast to wild-type mice, MME-/- mice did not have increased numbers of macrophages in their lungs and did not develop emphysema in response to long-term exposure to cigarette smoke. Smoke-exposed MME-/- mice that received monthly intratracheal instillations of monocyte chemoattractant protein-1 showed accumulation of alveolar macrophages but did not develop air space enlargement. Thus, macrophage elastase is probably sufficient for the development of emphysema that results from chronic inhalation of cigarette smoke.

Animals↗

PubMed1997

Elastin degradation by matrix metalloproteinases. Cleavage site specificity and mechanisms of elastolysis.

Insoluble elastin was used as a substrate to characterize the peptide bond specificities of human (HME) and mouse macrophage elastase (MME) and to compare these enzymes with other mammalian metalloproteinases and serine elastases. New amino termini detected by protein sequence analysis in insoluble elastin following proteolytic digestion reveal the P'1 residues in the carboxyl-terminal direction from the scissile bond. The relative proportion of each amino acid in this position reflects the proteolytic preference of the elastolytic enzyme. The predominant amino acids detected by protein sequence analysis following cleavage of insoluble elastin with HME, MME, and 92-kDa gelatinase were Leu, Ile, Ala, Gly, and Val. HME and MME were similar in their substrate specificity and showed a stronger preference for Leu/Ile than did the 92-kDa enzyme. Fibroblast collagenase showed no activity toward elastin. The amino acid residues detected in insoluble elastin following hydrolysis with porcine pancreatic elastase and human neutrophil elastase were predominantly Gly and Ala, with lesser amounts of Val, Phe, Ile, and Leu. There were interesting specificity differences between the two enzymes, however. For both the serine and matrix metalloproteinases, catalysis of peptide bond cleavage in insoluble elastin was characterized by temperature effects and water requirements typical of common enzyme-catalyzed reactions, even those involving soluble substrates. In contrast to what has been observed for collagen, the energy requirements for elastolysis were not extraordinary, consistent with cleavage sites in elastin being readily accessible to enzymatic attack.

Amino Acid Sequence↗

PubMed1997

Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase.

Macrophage elastase (ME) was originally named when metal-dependent elastolytic activity was detected in conditioned media of murine macrophages. Subsequent cDNA cloning of the mouse and human enzyme demonstrated that ME is a distinct member of the matrix metalloproteinase family. To date, the catalytic parameters that describe the hydrolysis of elastin by ME have not been quantified and its activity against other matrix proteins have not been described. In this report, we have examined the action of purified recombinant human ME (rHME), produced in Escherichia coli, on elastin and other extracellular matrix proteins. On a molar basis, rHME is approximately 30% as active as human leukocyte elastase in solubilizing elastin. rHME also efficiently degrades alpha1-antitrypsin (alpha1-AT), the primary physiological inhibitor of human leukocyte elastase. In addition, rHME efficiently degrades fibronectin, laminin, entactin, type IV collagen, chondroitan sulfate, and heparan sulfate. These results suggest that HME may be required for macrophages to penetrate basement membranes and remodel injured tissue during inflammation. Moreover, abnormal expression of HME may contribute to destructive processes such as pulmonary emphysema and vascular aneurysm formation. To further understand the specificity of HME, the initial cleavage sites in alpha1-AT have been determined. In addition, the hydrolysis of a series of synthetic peptides with different P'1 residues has been determined. rHME can accept large and small amino acids at the P'1 site, but has a preference for leucine.

Amino Acid Sequence↗

PubMed1997

Characterization of the mouse neutrophil elastase gene and localization to chromosome 10.

Neutrophil elastase (NE), a serine proteinase, is considered to play a role in normal tissue turnover and host defense, NE may also cause tissue damage in acute and chronic inflammatory diseases. We have isolated and characterized the gene for mouse NE and determined its chromosomal location. The mouse NE gene has been localized by interspecific backcross analysis to Chromosome (Chr) 10. The gene for mouse NE is composed of 5 exons and 4 introns, similar to the human NE. Mouse NE shares the highly conserved exon size and intron-exon borders with human NE. The coding exons of the mouse NE gene predict a translation product in a pre-pro form, similar to human NE. Knowledge of the genomic organization and chromosomal location of mouse NE may allow us to further define mechanisms responsible for cell and tissue-specific expression of mouse NE.

Animals↗

PubMed1997

Mighty mice: transgenic technology "knocks out" questions of matrix metalloproteinase function.

Matrix metalloproteinases (MMP) comprise a family of structurally related proteinases that are believed to play a critical role in many physiological and pathological processes. Transgenic technology offers the possibility of determining whether MMPs contribute directly to these processes. For example, gain of function and loss of function models have confirmed causative roles of MMPs in the development of pulmonary emphysema and unexpectedly uncovered an MMP-dependent mechanism of inflammatory cell recruitment. Limitations of these techniques and powerful applications on the horizon are also presented as we embark on an era where controlled experiments can be performed in complex mammalian models.

Animals↗

PubMed1997

A randomized, controlled trial of protocol-directed versus physician-directed weaning from mechanical ventilation.

OBJECTIVE: To compare a practice of protocol-directed weaning from mechanical ventilation implemented by nurses and respiratory therapists with traditional physician-directed weaning. DESIGN: Randomized, controlled trial. SETTING: Medical and surgical intensive care units in two university-affiliated teaching hospitals. PATIENTS: Patients requiring mechanical ventilation (n = 357). INTERVENTIONS: Patients were randomly assigned to receive either protocol-directed (n = 179) or physician-directed (n = 178) weaning from mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: The primary outcome measure was the duration of mechanical ventilation from tracheal intubation until discontinuation of mechanical ventilation. Other outcome measures included need for reintubation, length of hospital stay, hospital mortality rate, and hospital costs. The median duration of mechanical ventilation was 35 hrs for the protocol-directed group (first quartile 15 hrs; third quartile 114 hrs) compared with 44 hrs for the physician-directed group (first quartile 21 hrs; third quartile 209 hrs). Kaplan-Meier analysis demonstrated that patients randomized to protocol-directed weaning had significantly shorter durations of mechanical ventilation compared with patients randomized to physician-directed weaning (chi 2 = 3.62, p = .057, log-rank test; chi 2 = 5.12, p = .024, Wilcoxon test). Cox proportional-hazards regression analysis, adjusting for other covariates, showed that the rate of successful weaning was significantly greater for patients receiving protocol-directed weaning compared with patients receiving physician-directed weaning (risk ratio 1.31; 95% confidence interval 1.15 to 1.50; p = .039). The hospital mortality rates for the two treatment groups were similar (protocol-directed 22.3% vs. physician-directed 23.6%; p = .779). Hospital cost savings for patients in the protocol-directed group were $42,960 compared with hospital costs for patients in the physician-directed group. CONCLUSION: Protocol-guided weaning of mechanical ventilation, as performed by nurses and respiratory therapists, is safe and led to extubation more rapidly than physician-directed weaning.

Adult↗

PubMed1997

Mechanical ventilation with or without daily changes of in-line suction catheters.

The purpose of this study was to determine the safety and cost-effectiveness of not routinely changing in-line suction catheters for patients requiring mechanical ventilation. Patients were randomly assigned to receive either no routine in-line suction catheter changes (n = 258) or in-line suction catheter changes every 24 h (n = 263). The main outcome measure was the incidence of ventilator-associated pneumonia. Other outcomes evaluated included hospital mortality, acquired organ system derangements, duration of mechanical ventilation, lengths of intensive care and hospital stay, and the cost for in-line suction catheters. Ventilator-associated pneumonia was seen in 38 patients (14.7%) receiving no routine in-line suction catheter changes and in 39 patients (14.8%) receiving in-line suction catheter changes every 24 h (relative risk, 0.99; 95% CI, 0.66 to 1.50). No statistically significant differences for hospital mortality, lengths of stay, the number of acquired organ system derangements, death in patients with ventilator-associated pneumonia, or mortality directly attributed to ventilator-associated pneumonia were found between the two treatment groups. Patients receiving in-line suction catheter changes every 24 h had 1,224 catheter changes costing a total of $11,016; patients receiving no routine in-line suction catheter changes had a total of 93 catheter changes costing $837. Our findings suggest that the elimination of routine in-line suction catheter changes is safe and can reduce the costs associated with providing mechanical ventilation.

Adult↗

PubMed1997

Patient transport from intensive care increases the risk of developing ventilator-associated pneumonia.

STUDY OBJECTIVE: To determine whether patient transport out of the ICU is associated with an increased risk of developing ventilator-associated pneumonia. DESIGN: Prospective cohort study. SETTING: ICUs of Barnes-Jewish Hospital, a university-affiliated teaching hospital. PATIENTS: Five hundred twenty-one ICU patients requiring mechanical ventilation for > 12 h. INTERVENTION: Prospective patient surveillance and data collection. MEASUREMENTS AND RESULTS: The primary outcome measure was the development of ventilator-associated pneumonia. A total of 273 (52.4%) mechanically ventilated patients required at least one transport out of the ICU while 248 (47.6%) patients did not undergo transport. Sixty-six (24.2%) of the transported patients developed ventilator-associated pneumonia compared with 11 (4.4%) patients in the group not undergoing transport (relative risk=5.5; 95% confidence interval [CI]=2.9 to 10.1; p<0.001). Multiple logistic regression analysis demonstrated that a preceding episode of transport out of the ICU was independently associated with the development of ventilator-associated pneumonia (adjusted odds ratio=3.8; 95% CI=2.6 to 5.5; p<0.001). Other variables independently associated with the development of ventilator-associated pneumonia included reintubation, presence of a tracheostomy, administration of aerosols, and male gender. CONCLUSIONS: We conclude that patient transport out of the ICU is associated with an increased risk for the development of ventilator-associated pneumonia.

APACHE↗

PubMed1996

Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

Antigens, CD↗

PubMed1996

Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice.

Macrophages secrete a variety of proteinases that are thought to participate in remodeling of the extracellular matrix associated with inflammatory processes. We have eliminated expression of the macrophage metalloelastase (MME) gene by targeted disruption to assess the role of this protein in macrophage-mediated proteolysis. We found that the macrophages of MME-deficient (MME-/-) mice have a markedly diminished capacity to degrade extracellular matrix components. In addition, MME-/- macrophages are essentially unable to penetrate reconstituted basement membranes in vitro and in vivo. MME is therefore required for macrophage-mediated extracellular matrix proteolysis and tissue invasion.

Animals↗

PubMed1996

The structural basis for the elastolytic activity of the 92-kDa and 72-kDa gelatinases. Role of the fibronectin type II-like repeats.

Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. To test the role of the fibronectin type II-like repeats in elastolytic activity, we expressed the 92-kDa gelatinase CD with its fibronectin type II-like repeats (92 CD/FN) in yeast. 92 CD/FN degraded insoluble elastin with activity comparable to full-length 92-kDa gelatinase. 92 and 72 CDs lacking the fibronectin type II-like repeats did not bind elastin, whereas the parent enzymes and 92 CD/FN did bind elastin. Furthermore, recombinant 92-kDa fibronectin type II-like repeats inhibited binding of the 92-kDa gelatinase to elastin. We conclude that the 92- and 72-kDa gelatinases require the fibronectin type II-like repeats for elastase activity.

Amino Acid Sequence↗

PubMed1995

Mechanical ventilation with or without 7-day circuit changes. A randomized controlled trial.

OBJECTIVE: To determine whether a practice of not routinely changing ventilator circuits in patients who require prolonged mechanical ventilation is associated with an increased incidence of nosocomial pneumonia. DESIGN: Randomized controlled trial. SETTING: Intensive care units in two university-affiliated teaching hospitals. PATIENTS: 300 patients admitted to an intensive care unit who required mechanical ventilation for more than 5 days. INTERVENTION: Patients were randomly assigned to receive either no routine ventilator circuit changes or circuit changes every 7 days. MEASUREMENTS: The primary outcome measure was the incidence of ventilator-associated pneumonia. Other outcome measures included duration of mechanical ventilation, length of hospital stay, and hospital mortality. RESULTS: 147 patients were randomly assigned to receive no routine ventilator circuit changes, and 153 patients were randomly assigned to receive circuit changes every 7 days. The two groups were similar at the time of randomization with regard to demographic characteristics, intensive care unit admission diagnoses, and severity of illness. Ventilator-associated pneumonia was seen in 36 patients (24.5%) receiving no routine changes and in 44 patients (28.8%) receiving changes every 7 days (relative risk, 0.85 [95% CI, 0.55 to 1.17]). No statistically significant differences for hospital mortality, intensive care unit mortality, death during mechanical ventilation, death in patients with ventilator-associated pneumonia, or mortality directly attributed to ventilator-associated pneumonia were found between the two treatment groups (P > or = 0.11). Patients receiving changes every 7 days had 247 circuit changes costing a total of $7410; patients receiving no routine changes had a total of 11 circuit changes costing $330. CONCLUSION: The elimination of routine ventilator circuit changes can reduce medical care costs without increasing the incidence of nosocomial pneumonia in patients who require prolonged mechanical ventilation.

Adult↗

PubMed1995

Human macrophage metalloelastase. Genomic organization, chromosomal location, gene linkage, and tissue-specific expression.

Human macrophage metalloelastase (HME) is a recent addition to the matrix metalloproteinase (MMP) family that was initially found to be expressed in alveolar macrophages of cigarette smokers. To understand more about HME expression, analysis of the structure and location of the gene was performed. The gene for HME is composed of 10 exons and 9 introns, similar to the stromelysins and collagenases, and HME shares the highly conserved exon size and intron-exon borders with other MMPs. The 13-kilobase (kb) HME gene has been localized by fluorescence in situ hybridization to chromosome 11q22.2-22.3, the same location of the interstitial collagenase and stromelysin genes. We determined that HME and stromelysin 1 genes are physically linked within 62 kb utilizing pulse-field gel electrophoresis. The promoter region of the HME gene contains several features common to other MMP genes including a TATA box 29 bp upstream to the transcription initiation site, an AP-1 motif, and a PEA3 element. HME mRNA is not detectable in normal adult tissues but is induced in rapidly remodeling tissues such as the term placenta. In situ hybridization and immunohistochemistry of placental tissue demonstrated HME mRNA and protein expression in macrophages and stromal cells. Cell-specific expression and response to inflammatory stimuli such as endotoxin is conferred within 2.8 kb of the HME 5'-flanking sequence as demonstrated by HME promoter-CAT expression constructs. Knowledge of the genomic organization and chromosomal location of HME may allow us to further define mechanisms responsible for cell- and tissue-specific expression of HME.

Base Sequence↗

PubMed1995

Activation of the 92-kDa gelatinase by stromelysin and 4-aminophenylmercuric acetate. Differential processing and stabilization of the carboxyl-terminal domain by tissue inhibitor of metalloproteinases (TIMP).

The matrix metalloproteinase 92-kDa gelatinase is a major product of inflammatory cells. Macrophages synthesize and secrete this proteinase as a proenzyme in association with tissue inhibitor of metalloproteinases (TIMP) (92TIMP), whereas neutrophils store and release it from secondary granules as a TIMP-free proenzyme (92TIMP-free). Metalloproteinase proenzymes can be activated in vitro by a variety of agents, including organomercurials and proteinases, resulting in loss of an 8-10-kDa NH2-terminal domain which disrupts the interaction of a conserved cysteine residue with the catalytic zinc molecule. We report that the activation and processing of 92-kDa gelatinase differs depending on its association with TIMP and the nature of the activating agent. We observed that 92TIMP undergoes classic activation to 82 kDa by stromelysin, whereas exposure to 4-aminophenylmercuric acetate (APMA) results in a final product of 83 kDa that still contains the "prodomain" cysteine. Association with TIMP appears to stabilize the COOH-terminal domain, whereas 92TIMP-free is converted by APMA to a final product of 67 kDa lacking the COOH-terminal portion. In the continued presence of APMA, which maintains cysteine-zinc disruption, the 67-kDa species is at least as active as the classic 82 kDa. In contrast, activation of 92TIMP-free by stromelysin initially generates the 82-kDa form which is followed by final conversion to a 50-kDa species that lacks the catalytic domain of the parent molecule. Therefore, although stromelysin activation of 92TIMP-free is initially efficient, the active 82-kDa form is short-lived and is replaced by an inactive 50-kDa product. This complex pattern of activation of the 92-kDa gelatinase may serve to restrict its proteolytic capacity following exposure to stromelysin and may serve to regulate proteinase activity in vivo.

Amino Acid Sequence↗

PubMed1995

Mouse macrophage metalloelastase expressed in bacteria absolutely requires zinc for activity.

Mouse macrophage metalloelastase was expressed in Escherichia coli. This recombinant enzyme (rMME) was present in the inclusion bodies that were solubilized in 7 M guanidine HCl. After removal of guanidine HCl, rMME was purified with a Q-Sepharose column. Degradation of [3H]elastin by rMME absolutely required Ca2+; the optimal Ca2+ concentration was 5 mM. NaCl stimulated the enzyme activity; maximal stimulation was obtained at 400 mM. The rMME activity was inhibited by metalloprotease inhibitors, but not by serine, aspartyl, or thiol protease inhibitors. Among the divalent cations tested, only Ba2+ and Sr2+ exhibited marginal stimulation of rMME activity in the absence of Ca2+. Cu2+, Zn2+, or Cd2+ strongly inhibited rMME activity with IC50 values between 68 and 180 microM, while Mg2+, Ba2+, Mn2+, Co2+, and Sr2+ had no effect. The requirement of Zn2+ for rMME activity was determined. Significant enzyme activity was present in rMME treated with EDTA followed by Q-Sepharose column chromatography. Only when the inclusion bodies were solubilized in the presence of 20 mM EDTA, did an enzyme preparation which was absolutely dependent on exogenous Zn2+ for activity result. The optimal Zn2+ concentration for rMME activation was 100 microM. These results indicate that Zn2+ is tightly bound to rMME.

Amino Acid Sequence↗

PubMed1995

Agonist-induced expression of tissue inhibitor of metalloproteinases and metalloproteinases by human macrophages is regulated by endogenous prostaglandin E2 synthesis.

The regulatory effect of endogenously synthesized eicosanoid metabolites on the expression of tissue inhibitor of metalloproteinases (TIMP), interstitial collagenase, and 92-kDa gelatinase by human macrophages was examined. TIMP and metalloproteinase production were stimulated with three agonists that produce distinct patterns of eicosanoid synthesis: lipopolysaccharide (10 micrograms/ml), denatured collagen (10 micrograms/ml), or zymosan (1 mg/ml). Indomethacin (3 micrograms/ml) or MK886 (3 microM), a specific inhibitor of 5-lipoxygenase, was used to examine the role of endogenous metabolites of arachidonic acid. Regardless of the agonist used, TIMP production by macrophages was inhibited 65% by indomethacin, synthesis of interstitial collagenase was reduced 70%, and expression of 92-kDa gelatinase was decreased 40%. In contrast, inhibition of leukotriene synthesis had no effect on metalloproteinase or TIMP production. The agonist-stimulated increase in TIMP and collagenase production was directly correlated to the cumulative prostaglandin E2 level induced by the agonist used. However, if response to an agonist was poor, the exogenous addition of prostaglandin E2 could not increase TIMP or collagenase production more than twofold, indicating an important permissive effect of the agonist on the regulation of each protein's expression. The mechanism of indomethacin inhibition of TIMP and collagenase production was studied by labeling the cells with [35S]-methionine and performing immunoprecipitation using specific antiserum. Indomethacin markedly inhibited the lipopolysaccharide-induced biosynthesis of both TIMP and collagenase. Northern analysis revealed parallel suppression of TIMP and collagenase steady-state mRNA levels by indomethacin, indicating pretranslational control. The regulation of inflammatory-cell TIMP and interstitial collagenase expression by prostaglandin E2 suggests that therapy inhibiting the cellular response to prostaglandins may be useful in cutaneous and systemic disease states involving macrophage-mediated connective-tissue destruction.

Adult↗

PubMed1995

The pathogenesis of emphysema: the elastase:antielastase hypothesis 30 years later.

Thirty years ago the concept of the elastase:antielastase hypothesis was introduced. Neutrophil elastase:A1PI balance was found to be critical, predisposing patients deficient in A1PI to panacinar emphysema; however, the causative elastases in common cigarette-induced pulmonary emphysema are unclear. Several members of the serine, cysteine, and metalloproteinase families have been identified in inflammatory and resident lung cells. We propose to use gene-targeted mice, deficient in individual elastases to determine the relative contribution of candidate elastases to cigarette-smoking-related emphysema. This does not guarantee that humans will respond to cigarette smoke with the same array of elastases as mice. However, these studies may guide labor intensive therapeutic trials with specific proteinase inhibitors and ultimately lead to rational therapy.

Animals↗