Buprenorphine alters microglia and astrocytes acutely following diffuse traumatic brain injury.

Traumatic brain injury (TBI) is a common phenomenon, accounting for significant cost and adverse health effects. While there is information about focal pathologies following TBI, knowledge of more diffuse processes is lacking, particularly regarding how analgesics affect this pathology. As buprenorphine is the most commonly used analgesic in experimental TBI models, this study investigated the acute effects of the opioid analgesic buprenorphine (Bup-SR-Lab) on diffuse neuronal/glial pathology, neuroinflammation, cell damage, and systemic physiology.

Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix.

Smooth muscle cells contribute to extracellular matrix remodeling during atherogenesis. De-differentiated, synthetic smooth muscle cells are involved in processes of migration, proliferation and changes in expression of extracellular matrix components, all of which contribute to loss of homeostasis accompanying atherogenesis. Elevated levels of acute phase proteins, including serum amyloid A (SAA), are associated with an increased risk for atherosclerosis.

Trafficking of endogenous smooth muscle cell cholesterol: a role for serum amyloid A and interleukin-1β.

Objective: Intracellular cholesterol distribution impacts cell function; however, processes influencing endogenous cholesterol trafficking remain largely unknown. Atherosclerosis is associated with vascular inflammation and these studies address the role of inflammatory mediators on smooth muscle cell cholesterol trafficking.

Methods And Results: Interestingly, in the absence of an exogenous cholesterol source, serum amyloid A increased [(14)C] oleic acid incorporation into cholesteryl ester in rat smooth muscle cells, suggesting endogenous cholesterol trafficking to the endoplasmic reticulum.

Micropatterned cell sheets with defined cell and extracellular matrix orientation exhibit anisotropic mechanical properties.

For an arterial replacement graft to be effective, it must possess the appropriate strength in order to withstand long-term hemodynamic stress without failure, yet be compliant enough that the mismatch between the stiffness of the graft and the native vessel wall is minimized. The native vessel wall is a structurally complex tissue characterized by circumferentially oriented collagen fibers/cells and lamellar elastin. Besides the biochemical composition, the functional properties of the wall, including stiffness, depend critically on the structural organization.

A human surfactant peptide-elastase inhibitor construct as a treatment for emphysema.

Two million Americans suffer from pulmonary emphysema, costing $2.5 billion/year and contributing to 100,000 deaths/year. Emphysema is thought to result from an imbalance between elastase and endogenous inhibitors of elastase, leading to tissue destruction and a loss of alveoli.

The critical role of the cellular thiol homeostasis in cadmium perturbation of the lung extracellular matrix.

Cadmium (Cd) inhalation can result in emphysema. Cd exposure of rat lung fibroblasts (RFL6) enhanced levels of metal scavenging thiols, e.g.

A zipper network model of the failure mechanics of extracellular matrices.

Mechanical failure of soft tissues is characteristic of life-threatening diseases, including capillary stress failure, pulmonary emphysema, and vessel wall aneurysms. Failure occurs when mechanical forces are sufficiently high to rupture the enzymatically weakened extracellular matrix (ECM). Elastin, an important structural ECM protein, is known to stretch beyond 200% strain before failing.

Mechanical and failure properties of extracellular matrix sheets as a function of structural protein composition.

The goal of this study was to determine how alterations in protein composition of the extracellular matrix (ECM) affect its functional properties. To achieve this, we investigated the changes in the mechanical and failure properties of ECM sheets generated by neonatal rat aortic smooth muscle cells engineered to contain varying amounts of collagen and elastin. Samples underwent static and dynamic mechanical measurements before, during, and after 30 min of elastase digestion followed by a failure test.

Cloning and characterization of the rat lysyl oxidase gene promoter: identification of core promoter elements and functional nuclear factor I-binding sites.

Lysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5'-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from -78 to -51 upstream of ATG.

Perturbation of copper (Cu) homeostasis and expression of Cu-binding proteins in cadmium-resistant lung fibroblasts.

To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker.

Differential effects of static and cyclic stretching during elastase digestion on the mechanical properties of extracellular matrices.

Enzyme activity plays an essential role in many physiological processes and diseases such as pulmonary emphysema. While the lung is constantly exposed to cyclic stretching, the effects of stretch on the mechanical properties of the extracellular matrix (ECM) during digestion have not been determined. We measured the mechanical and failure properties of elastin-rich ECM sheets loaded with static or cyclic uniaxial stretch (40% peak strain) during elastase digestion.

New insights into the inhibition of human neutrophil elastase by heparin.

In the normal feedback mechanism of injury and repair in the lung, fragmented heparan sulfate proteoglycans (HSPGs) from damaged extracellular matrix and cells are believed to interact with elastases to limit their activity. An imbalance in the HSPG-elastase response may play an important role in situations where uncontrolled lung injury leads to diseases such as emphysema. To gain insight into this complex process of heparin and heparan sulfate regulation of elastases, an experimental study was undertaken to resolve the mechanism and structural requirements of heparin inhibition of human neutrophil elastase (HNE).

Crosslinked alpha-elastin biomaterials: towards a processable elastin mimetic scaffold.

Elastin is a critical biochemical and biomechanical component of vascular tissue. However, elastin is also highly insoluble and therefore difficult to process into new biomaterials. We present a simple approach for synthesizing elastin-based materials from two commercially available and water-soluble components: alpha-elastin and a diepoxy crosslinker.

Neutrophil elastase-initiated EGFR/MEK/ERK signaling counteracts stabilizing effect of autocrine TGF-beta on tropoelastin mRNA in lung fibroblasts.

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938-18946, 2002).

Inhibition of the expression of lysyl oxidase and its substrates in cadmium-resistant rat fetal lung fibroblasts.

Copper (Cu)-dependent lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin stabilizing the extracellular matrix (ECM). Chronic inhalation of cadmium (Cd), a toxic metal, induces emphysema. To probe mechanisms of Cd injury to the lung, we developed Cd-resistant (CdR) cells from rat fetal lung fibroblasts (RFL6) by chronic exposure to CdCl(2) from 1 to 40 microM and further examined their expressions of LO, LO substrates, and Cu-scavenging thiols.

Hypoxia suppresses elastin repair by rat lung fibroblasts.

Macrophage and neutrophil proteinases damage lung elastin, disrupting alveolar epithelium and filling alveoli with inflammatory exudate. Alveolar collapse and regional hypoxia occur. Whether low oxygen tension alters fibroblast-mediated lung repair is unknown.

Transcriptional and posttranscriptional inhibition of lysyl oxidase expression by cigarette smoke condensate in cultured rat fetal lung fibroblasts.

Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0-120 microg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80-120 microg CSC/ml.

Effects of elastase on the mechanical and failure properties of engineered elastin-rich matrices.

Pulmonary emphysema and vessel wall aneurysms are diseases characterized by elastolytic damage to elastin fibers that leads to mechanical failure. To model this, neonatal rat aortic smooth muscle cells were cultured, accumulating an extracellular matrix rich in elastin, and mechanical measurements were made before and during enzymatic digestion of elastin. Specifically, the cells in the cultures were killed with sodium azide, the cultures were lifted from the flask, cut into small strips, and fixed to a computer-controlled lever arm and a force transducer.

B-Myb represses elastin gene expression in aortic smooth muscle cells.

B-Myb represses collagen gene transcription in vascular smooth muscle cells (SMCs) in vitro and in vivo. Here we sought to determine whether elastin is similarly repressed by B-Myb. Levels of tropoelastin mRNA and protein were lower in aortas and isolated SMCs of adult transgenic mice expressing the human B-myb gene, driven by the basal cytomegalovirus promoter, compared with age-matched wild type (WT) animals.

Downregulation of lysyl oxidase and upregulation of cellular thiols in rat fetal lung fibroblasts treated with cigarette smoke condensate.

Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e.

A(3) adenosine receptor deficiency does not influence atherogenesis.

Atherosclerosis is a multifactorial disease, the progression of which is modulated by several factors, including inflammation and hypercholesterolemia. The A(3) adenosine receptor (A(3)AR) has been reported to affect mast cell degranulation leading to inflammation, as well as to influence cardiovascular homeostasis. Here, we show that its deletion can also impact vascular smooth muscle cell (VSMC) proliferation in vitro.

B-Myb represses vascular smooth muscle cell collagen gene expression and inhibits neointima formation after arterial injury.

Objective: The function of B-Myb, a negative regulator of vascular smooth muscle cell (SMC) matrix gene transcription, was analyzed in the vasculature.

Methods And Results: Mice were generated in which the human B-myb gene was driven by the basal cytomegalovirus promoter, and 3 founders were identified. Mice appeared to develop normally, and human B-myb was expressed in the aortas.

Elastase mediates the release of growth factors from lung in vivo.

Uncontrolled elastase activity is involved in the development of several types of lung disease. Previous reports demonstrated that growth factors are liberated from pulmonary matrix storage sites by elastase; however, release of these entities in vivo is not well defined. In the present study, we investigated the release of fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-beta), after intratracheal instillation of porcine pancreatic elastase into mice.

Transcriptional regulation of pulmonary elastin gene expression in elastase-induced injury.

Previously we have shown that treatment of confluent, pulmonary fibroblast cultures with elastase results in upregulation of elastin mRNA and protein levels. In the present study we focused on determining the level at which elastin expression is upregulated after elastase exposure. We examined as models for this investigation elastin gene expression in primary pulmonary fibroblast cells during the transition from subconfluent to confluent cultures and in confluent, matrix-laden cultures treated briefly with elastase.

Heparan sulfate depletion within pulmonary fibroblasts: implications for elastogenesis and repair.

We investigated the role of sulfated proteoglycans in regulating extracellular matrix (ECM) deposition in pulmonary fibroblast cultures. Fibroblast cultures were subject to pharmacologic and enzymatic interventions to modify sulfated proteoglycan levels. Native and proteoglycan-depleted fibroblasts were treated with porcine pancreatic elastase at 2-4-day intervals and the elastase-mediated release of fibroblast growth factor 2 (FGF-2) and glycosaminoglycans was determined.