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Biomedical subjects

S D Shapiro

Publications and source records attributed to S D Shapiro.

At least 37 records · Page 2Linked to original sources
PubMed2000

Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium.

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1-negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2'-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with beta-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.

Animals↗
PubMed2000

Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase.

In determining the mechanism of neutrophil elastase (NE)-mediated killing of Escherichia coli, we found that NE degraded outer membrane protein A (OmpA), localized on the surface of Gram-negative bacteria. NE killed wild-type, but not OmpA-deficient, E. coli. Also, whereas NE-deficient mice had impaired survival in response to E. coli sepsis, as compared to wild-type mice, the presence or absence of NE had no influence on survival in response to sepsis that had been induced with OmpA-deficient E. coli. These findings define a mechanism of nonoxidative bacterial killing by NE and point to OmpA as a bacterial target in host defense.

Animals↗
PubMed2000

Evolving concepts in the pathogenesis of chronic obstructive pulmonary disease.

It is arguable that more biologic insight has been gained from the study of COPD than from any other pulmonary disorder. A vast knowledge of the biology of extracellular matrix proteins, proteinases, and proteinase inhibitors has largely stemmed from the elastase:anti-elastase hypothesis for the pathogenesis of emphysema. An equally compelling case could be made that interest in, and funding for, COPD research has been woeful, and investigators have made no significant medical breakthroughs in the treatment of this disorder, which, unfortunately, is becoming epidemic worldwide. Indeed, it cannot be argued that physicians have very little treatment to offer to the many patients with COPD. Humankind is rapidly approaching a time when all human genes will be sequenced, and genetic engineering will allow determination of the function of these proteins in vivo. Expression profiling and bioinformatics will allow clinicians to assess the spectrum of genes and proteins regulated in biologic processes, no longer limiting study to naïve candidate genes. These advances will allow investigators to decipher precise pathways of complex diseases, identify genetic and environmental interactions, and ultimately lead to specific (pre)diagnoses and rational treatment. Answers to the question as to why only a subset of smokers develop COPD will enhance the understanding of the disease process. Fortunately, there has been a resurgence of interest in COPD, led largely by the pharmaceutical industry, which has discovered the potential of this unmet need. Consequently, these state-of-the-art scientific techniques are being directly applied to COPD, lending hope for the future. Of even greater importance, the tide seems to be turning on the cigarette industry. Although difficult to imagine, perhaps cigarettes will disappear in this lifetime or at least the next generation won't be fooled by this deadly habit! Well ... the rational therapy thing could work.

Airway Obstruction↗
PubMed2000

Expression of human macrophage metalloelastase (MMP-12) by tumor cells in skin cancer.

Matrix metalloproteinases play an essential role in tumor growth and invasion. Different matrix metalloproteinases are often expressed in cancers with distinct patterns. To investigate the role of human macrophage metalloelastase (MMP-12) in epidermal tumors, we studied human macrophage metalloelastase mRNA and protein expression in malignant squamous cell and basal cell carcinomas, and in premalignant Bowen's disease. Human macrophage metalloelastase was detected in 11 of 17 squamous cell carcinomas in epithelial cancer cells, whereas macrophages were positive in 15 of 17 samples. In basal cell carcinomas, human macrophage metalloelastase was more often found in macrophages (seven of 19) than in cancer cells (four of 19). Human macrophage metalloelastase mRNA was also detected in three cell lines derived from squamous cell carcinomas of the head and neck and in transformed HaCaT cells, whereas premalignant tumors and primary keratinocytes were negative for human macrophage metalloelastase mRNA. Western analysis revealed human macrophage metalloelastase protein in squamous cell carcinoma cells. Our results show that human macrophage metalloelastase can be expressed in vivo and in vitro by transformed epithelial cells and indicate that the level of human macrophage metalloelastase expression correlates with epithelial dedifferentiation and histologic aggressiveness.

Blotting, Northern↗
PubMed2000

Developmental expression of latent transforming growth factor beta binding protein 2 and its requirement early in mouse development.

Latent transforming growth factor beta (TGF-beta) binding protein 2 (LTBP-2) is an integral component of elastin-containing microfibrils. We studied the expression of LTBP-2 in the developing mouse and rat by in situ hybridization, using tropoelastin expression as a marker of tissues participating in elastic fiber formation. LTBP-2 colocalized with tropoelastin within the perichondrium, lung, dermis, large arterial vessels, epicardium, pericardium, and heart valves at various stages of rodent embryonic development. Both LTBP-2 and tropoelastin expression were seen throughout the lung parenchyma and within the cortex of the spleen in the young adult mouse. In the testes, LTBP-2 expression was seen within lumenal cells of the epididymis in the absence of tropoelastin. Collectively, these results imply that LTBP-2 plays a structural role within elastic fibers in most cases. To investigate its importance in development, mice with a targeted disruption of the Ltbp2 gene were generated. Ltbp2(-/-) mice die between embryonic day 3.5 (E3.5) and E6.5. LTBP-2 expression was not detected by in situ hybridization in E6.5 embryos but was detected in E3.5 blastocysts by reverse transcription-PCR. These results are not consistent with the phenotypes of TGF-beta knockout mice or mice with knockouts of other elastic fiber proteins, implying that LTBP-2 performs a yet undiscovered function in early development, perhaps in implantation.

Animals↗
PubMed2000

Substance P and neurokinin-1 receptor expression by intrinsic airway neurons in the rat.

Tachykinins and their receptors are involved in the amplification of inflammation in the airways. We analyzed the expression of preprotachykinin-A (PPT-A) and neurokinin-1 (NK-1) receptor genes by intrinsic airway neurons in the rat. We also tested the hypothesis that PPT-A-encoded peptides released by these neurons fulfill the requisite role of substance P in immune complex injury of the lungs. We found that ganglion neurons in intact and denervated airways or in primary culture coexpress PPT-A and NK-1 receptor mRNAs and their protein products. Denervated ganglia from tracheal xenografts (nu/nu mice) or syngeneic lung grafts had increased PPT-A mRNA contents, suggesting preganglionic regulation. Formation of immune complexes in the airways induced comparable inflammatory injuries in syngeneic lung grafts, which lack peptidergic sensory fibers, and control lungs. The injury was attenuated in both cases by pretreatment with the NK-1 receptor antagonist LY-306740. We conclude that tachykinins released by ganglia act as a paracrine or autocrine signal in the airways and may contribute to NK-1 receptor-mediated amplification of immune injury in the lungs.

Acetamides↗
PubMed2000

Effect of partial liquid ventilation on pulmonary vascular permeability and edema after experimental acute lung injury.

We evaluated the effects of partial liquid ventilation (PLV) with two different dosages of the perfluorocarbon LiquiVent (perflubron) on pulmonary vascular permeability and edema formation after oleic acid (OA)-induced acute lung injury in dogs. We used imaging with positron emission tomography to measure fractional pulmonary blood flow, lung water concentration (LWC), and the pulmonary transcapillary escape rate (PTCER) of (68)Ga-labeled transferrin at 5 and 21 h after lung injury in five dogs undergoing conventional mechanical ventilation (CMV), five dogs undergoing low-dose PLV (perflubron at 10 ml/kg), and four dogs undergoing high dose PLV (perflubron at 30 ml/kg). A positive end-expiratory pressure of 7.5 cm H(2)O was used in all dogs. After OA (0.08 ml/kg)- induced lung injury, there were no significant differences or trends for PTCER or LWC at any time when the PLV groups were compared with the CMV group. However, lung tissue myeloperoxidase activity was significantly lower in the combined PLV group than in the CMV group (p = 0.016). We conclude that after OA-induced lung injury, the addition of PLV to CMV does not directly attenuate pulmonary vascular leak or lung water accumulation. Rather, the benefits of such treatment may be due to modifications of the inflammatory response.

Animals↗
PubMed2000

Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice.

Cilia have been classified as sensory or motile types on the basis of functional and structural characteristics; however, factors important for regulation of assembly of different cilia types are not well understood. Hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) is a winged helix/forkhead transcription factor expressed in ciliated cells of the respiratory tract, oviduct, and ependyma in late development through adulthood. Targeted deletion of the Hfh4 gene resulted in defective ciliogenesis in airway epithelial cells and randomized left-right asymmetry so that half the mice had situs inversus. In HFH-4-null mice, classic motile type cilia with a 9 + 2 microtubule ultrastructure were absent in epithelial cells, including those in the airways. In other organs, sensory cilia with a 9 + 0 microtubule pattern, such as those on olfactory neuroepithelial cells, were present. Ultrastructural analysis of mutant cells with absent 9 + 2 cilia demonstrated that defective ciliogenesis was due to abnormal centriole migration and/or apical membrane docking, suggesting that HFH-4 functions to direct basal body positioning or anchoring. Evaluation of wild-type embryos at gestational days 7.0 to 7.5 revealed Hfh4 expression in embryonic node cells that have monocilium, consistent with a function for this factor at the node in early determination of left- right axis. Analysis of the node of HFH-4 mutant embryos revealed that, in contrast to absent airway cilia, node cilia were present. These observations indicate that there are independent regulatory pathways for node ciliogenesis compared with 9 + 2 type ciliogenesis in airways, and support a central role for HFH-4 in ciliogenesis and left-right axis formation.

Abnormalities, Multiple↗
PubMed2000

Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema.

Cigarette smoke exposure is the major cause of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop significant COPD, and patients with asthma or asthma-like airway hyperresponsiveness or eosinophilia experience accelerated loss of lung function after cigarette smoke exposure. Pulmonary inflammation is a characteristic feature of lungs from patients with COPD. Surprisingly, the mediators of this inflammation and their contributions to the pathogenesis and varied natural history of COPD are not well defined. Here we show that IL-13, a critical cytokine in asthma, causes emphysema with enhanced lung volumes and compliance, mucus metaplasia, and inflammation, when inducibly overexpressed in the adult murine lung. MMP-2, -9, -12, -13, and -14 and cathepsins B, S, L, H, and K were induced by IL-13 in this setting. In addition, treatment with MMP or cysteine proteinase antagonists significantly decreased the emphysema and inflammation, but not the mucus in these animals. These studies demonstrate that IL-13 is a potent stimulator of MMP and cathepsin-based proteolytic pathways in the lung. They also demonstrate that IL-13 causes emphysema via a MMP- and cathepsin-dependent mechanism(s) and highlight common mechanisms that may underlie COPD and asthma.

Animals↗
PubMed2000

A critical role for neutrophil elastase in experimental bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune skin disease characterized by subepidermal blisters and autoantibodies against 2 hemidesmosome-associated proteins, BP180 and BP230. The immunopathologic features of BP can be reproduced in mice by passive transfer of anti-BP180 antibodies. Lesion formation in this animal model depends upon complement activation and neutrophil recruitment. In the present study, we investigated the role of neutrophil elastase (NE) in antibody-induced blister formation in experimental BP. Abnormally high levels of caseinolytic activity, consistent with NE, were detected in extracts of lesional skin and blister fluid of mice injected with anti-BP180 IgG. The pathogenic anti-BP180 IgG failed to induce subepidermal blistering in NE-null (NE(-/-)) mutant mice. NE(-/-) mice reconstituted with neutrophils from wild-type mice became susceptible to experimental BP. Wild-type mice given NE inhibitors (alpha1-proteinase inhibitor and Me-O-Suc-Ala-Ala-Pro-Val-CH(2)Cl), but not mice given cathepsin G/chymase inhibitors (alpha1-antichymotrypsin or Z-Gly-Leu-Phe-CH(2)Cl), were resistant to the pathogenic activity of anti-BP180 antibodies. Incubation of murine skin with NE induced BP-like epidermal-dermal detachment. Finally, NE cleaved BP180 in vitro and in vivo. These results implicate NE directly in the dermal-epidermal cleavage induced by anti-BP180 antibodies in the experimental BP model.

Animals↗
PubMed2000

Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.

Abdominal aortic aneurysms represent a life-threatening condition characterized by chronic inflammation, destructive remodeling of the extracellular matrix, and increased local expression of matrix metalloproteinases (MMPs). Both 92-kD gelatinase (MMP-9) and macrophage elastase (MMP-12) have been implicated in this disease, but it is not known if either is necessary in aneurysmal degeneration. We show here that transient elastase perfusion of the mouse aorta results in delayed aneurysm development that is temporally associated with transmural mononuclear inflammation, increased local production of several elastolytic MMPs, and progressive destruction of the elastic lamellae. Elastase-induced aneurysmal degeneration was suppressed by treatment with a nonselective MMP inhibitor (doxycycline) and by targeted gene disruption of MMP-9, but not by isolated deficiency of MMP-12. Bone marrow transplantation from wild-type mice prevented the aneurysm-resistant phenotype in MMP-9-deficient animals, and wild-type mice acquired aneurysm resistance after transplantation from MMP-9-deficient donors. These results demonstrate that inflammatory cell expression of MMP-9 plays a critical role in an experimental model of aortic aneurysm disease, suggesting that therapeutic strategies targeting MMP-9 may limit the growth of small abdominal aortic aneurysms.

Animals↗
PubMed2000

The effect of respiratory therapist-initiated treatment protocols on patient outcomes and resource utilization.

CONTEXT: Physicians frequently prescribe respiratory treatments to hospitalized patients, but the influence of such treatments on clinical outcomes is difficult to assess. OBJECTIVE: To compare the clinical outcomes of patients receiving respiratory treatments managed by respiratory care practitioner (RCP)-directed treatment protocols or physician-directed orders. DESIGN: A single center, quasi-randomized, clinical study. SETTING: Three internal medicine firms from an urban teaching hospital. PATIENTS: Six hundred ninety-four consecutive hospitalized non-ICU patients ordered to receive respiratory treatments. MAIN OUTCOME MEASURES: Discordant respiratory care orders, respiratory care charges, hospital length of stay, and patient-specific complications. Discordant orders were defined as written orders for respiratory treatments that were not clinically indicated as well as orders omitting treatments that were clinically indicated according to protocol-based treatment algorithms. RESULTS: Firm A patients (n = 239) received RCP-directed treatments and had a statistically lower rate of discordant respiratory care orders (24.3%) as compared with patients receiving physician-directed treatments in firms B (n = 205; 58.5%) and C (n = 250; 56.8%; p < 0.001). No statistically significant differences in patient complications were observed. The average number of respiratory treatments and respiratory care charges were statistically less for firm A patients (10.7 +/- 13.7 treatments; $868 +/- 1,519) as compared with patients in firms B (12.4 +/- 12.7 treatments, $1,124 +/- 1,339) and C (12.3 +/- 13.4 treatments, $1, 054 +/- 1,346; p = 0.009 [treatments] and p < 0.001 [respiratory care charges]). CONCLUSIONS: Respiratory care managed by RCP-directed treatment protocols for non-ICU patients is safe and showed greater agreement with institutional treatment algorithms as compared with physician-directed respiratory care. Additionally, the overall utilization of respiratory treatments was significantly less among patients receiving RCP-directed respiratory care.

Adult↗
PubMed1999

Normal neutrophil function in cathepsin G-deficient mice.

Cathepsin G is a neutral serine protease that is highly expressed at the promyelocyte stage of myeloid development. We have developed a homologous recombination strategy to create a loss-of-function mutation for murine cathepsin G. Bone marrow derived from mice homozygous for this mutation had no detectable cathepsin G protein or activity, indicating that no other protease in bone marrow cells has the same specificity. Hematopoiesis in cathepsin G-/- mice is normal, and the mice have no overt abnormalities in blood clotting. Neutrophils derived from cathepsin G-/- mice have normal morphology and azurophil granule composition; these neutrophils also display normal phagocytosis and superoxide production and have normal chemotactic responses to C5a, fMLP, and interleukin-8. Although cathepsin G has previously shown to have broad spectrum antibiotic properties, challenges of mice with Staphylococcus aureus, Klebsiella pneumoniae, or Escherichia coli yielded survivals that were not different from those of wild-type animals. In sum, cathepsin G-/- neutrophils have no obvious defects in function; either cathepsin G is not required for any of these normal neutrophil functions or related azurophil granule proteases with different specificities (ie, neutrophil elastase, proteinase 3, azurocidin, and/or others) can substitute for it in vivo.

Animals↗
PubMed1999

Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodelling.

Fibrillin is the principal structural component of the 10-12 nm diameter elastic microfibrils of the extracellular matrix. We have previously shown that both fibrillin molecules and assembled microfibrils are susceptible to degradation by serine proteases. In this study, we have investigated the potential catabolic effects of six matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-12, MMP-13 and MMP-14) on fibrillin molecules and on intact fibrillin-rich microfibrils isolated from ciliary zonules. Using newly synthesized recombinant fibrillin molecules, major cleavage sites within fibrillin-1 were identified. In particular, the six different MMPs generated a major degradation product of approximately 45 kDa from the N-terminal region of the molecule, whereas treatment of truncated, unprocessed and furin-processed C-termini also generated large degradation products. Introduction of a single ectopia lentis-causing amino acid substitution (E2447K; one-letter symbols for amino acids) in a calcium-binding epidermal growth factor-like domain, predicted to disrupt calcium binding, markedly altered the pattern of C-terminal fibrillin-1 degradation. However, the fragmentation pattern of a mutant fibrillin-1 with a comparable E-->K substitution in an upstream calcium-binding epidermal growth factor-like domain was indistinguishable from wild-type molecules. Ultrastructural examination highlighted that fibrillin-rich microfibrils isolated from ciliary zonules were grossly disrupted by MMPs. This is the first demonstration that fibrillin molecules and fibrillin-rich microfibrils are degraded by MMPs and that certain amino acid substitutions change the fragmentation patterns. These studies have important implications for physiological and pathological fibrillin catabolism and for loss of connective tissue elasticity in ageing and disease.

Aging↗
PubMed1999

Macrophage metalloelastase, MMP-12, cleaves human apolipoprotein(a) in the linker region between kringles IV-4 and IV-5. Potential relevance to lipoprotein(a) biology.

In this study we found that macrophage metalloelastase, MMP-12 cleaves, in vitro, apolipoprotein(a) (apo(a)) in the Asn3518-Val3519 bond located in the linker region between kringles IV-4 and IV-5, a bond immediately upstream of the Ile3520-Leu3521 bond, shown previously to be the site of action by neutrophil elastase (NE). We have also shown that human apo(a) injected into the tail vein of control mice undergoes degradation as reflected by the appearance of immunoreactive fragments in the plasma and in the urine of these animals. To define whether either or both of these enzymes may be responsible for the in vivo apo(a) cleavage, we injected intravenously MMP-12(-/-), NE -/- mice and litter mates, all of the same strain, with either lipoprotein(a) (Lp(a)), full-length free apo(a), or its N-terminal fragment, F1, obtained by the in vitro cleavage of apo(a) by NE. In the plasma of Lp(a)/apo(a)-injected mice, F1 was detected in control and NE -/- mice but was virtually absent in the MMP-12(-/-) mice. Moreover, fragments of the F1 type were present in the urine of the animals except for the MMP-12(-/-) mice. These fragments were significantly smaller in size than those observed in the plasma. All of the animals injected with F1 exhibited small sized fragments in their urine. These observations provide evidence that, in the mouse strain used, MMP-12 plays an important role in the generation of F1 from injected human Lp(a)/apo(a) and that this fragment undergoes further cleavage during renal transit via a mechanism that is neither NE- nor MMP-12-dependent. Thus, factors influencing the expression of MMP-12 may have a modulating action on the biology of Lp(a).

Amino Acid Sequence↗