Epithelial cells exert differential traction stress in response to substrate stiffness.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.

Hypoxia Induced Heparan Sulfate Primes the Extracellular Matrix for Endothelial Cell Recruitment by Facilitating VEGF-Fibronectin Interactions.

Vascular endothelial growth factor-A (VEGF) is critical for the development, growth, and survival of blood vessels. Retinal pigmented epithelial (RPE) cells are a major source of VEGF in the retina, with evidence that the extracellular matrix (ECM)-binding forms are particularly important. VEGF associates with fibronectin in the ECM to mediate distinct signals in endothelial cells that are required for full angiogenic activity. Hypoxia stimulates VEGF expression and angiogenesis; however, little is known about whether hypoxia also affects VEGF deposition within the ECM. Therefore, we investigated the role of hypoxia in modulating VEGF-ECM interactions using a primary retinal cell culture model. We found that retinal endothelial cell attachment to RPE cell layers was enhanced in cells maintained under hypoxic conditions. Furthermore, we found that agents that disrupt VEGF-fibronectin interactions inhibited endothelial cell attachment to RPE cells. We also found that hypoxia induced a general change in the chemical structure of the HS produced by the RPE cells, which correlated to changes in the deposition of VEGF in the ECM, and we further identified preferential binding of VEGFR2 over VEGFR1 to VEGF laden-fibronectin matrices. Collectively, these results indicate that hypoxia-induced HS may prime fibronectin for VEGF deposition and endothelial cell recruitment by promoting VEGF-VEGFR2 interactions as a potential means to control angiogenesis in the retina and other tissues.

Hypoxia modulates the development of a corneal stromal matrix model.

Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, we used 2 three-dimensional (3-D) models to examine how stromal cells respond to periods of acute hypoxic states. The first model, a stromal construct model, is a 3-D stroma-like construct that consists of human corneal fibroblasts (HCFs) stimulated by a stable form of ascorbate for 1, 2, and 4 weeks to self-assemble their own extracellular matrix. The second model, a corneal organ culture model, is a corneal wound-healing model, which consists of wounded adult rat corneas that were removed and placed in culture to heal. Both models were exposed to either normoxic or hypoxic conditions for varying time periods, and the expression and/or localization of matrix proteins was assessed. No significant changes were detected in Type V collagen, which is associated with Type I collagen fibrils; however, significant changes were detected in the expression of both the small leucine-rich repeating proteoglycans and the larger heparan sulfate proteoglycan, perlecan. Also, hypoxia decreased both the number of Cuprolinic blue-positive glycosaminoglycan chains along collagen fibrils and Sulfatase 1, which modulates the effect of heparan sulfate by removing the 6-O-sulfate groups. In the stromal construct model, alterations were seen in fibronectin, similar to those that occur in development and after injury. These changes in fibronectin after injury were accompanied by changes in proteoglycans. Together these findings indicate that acute hypoxic changes alter the physiology of the cornea, and these models will allow us to manipulate the conditions in the extracellular environment in order to study corneal development and trauma.

High fat diet induces pre-type 2 diabetes with regional changes in corneal sensory nerves and altered P2X7 expression and localization.

Type 2 diabetes is one of the leading pathologies that increases the risk of improper wound healing. Obesity has become a major risk factor for this disease that is now considered to be the 4th highest cause of preventable blindness according to the World Health Organization. The cornea is the most densely innervated structure in the human body and senses even the slightest injury. In diabetes, decreased corneal sensitivity secondary to diabetic peripheral neuropathy can lead to increased corneal abrasion, ulceration, and even blindness. In this study, a diet induced obesity (DIO) mouse model of pre-Type 2 diabetes was used to characterize changes in sensory nerves and P2X7, a purinoreceptor, a pain receptor, and an ion channel that is expressed in a number of tissues. Since our previous studies demonstrated that P2X7 mRNA was significantly elevated in diabetic human corneas, we examined P2X7 expression and localization in the DIO murine model at various times after being fed a high fat diet. Fifteen weeks after onset of diet, we found that there was a significant decrease in the density of sub-basal nerves in the DIO mice that was associated with an increase in tortuosity and a decrease in diameter. In addition, P2X7 mRNA expression was significantly greater in the corneal epithelium of DIO mice, and the increase in transcript was enhanced in the central migrating and peripheral regions after injury. Interestingly, confocal microscopy and thresholding analysis revealed that there was a significant increase in P2X7 distal to the injury, which contrasted with a decrease in P2X7-expressing stromal sensory nerves. Therefore, we hypothesize that the P2X7 receptor acts to sense changes at the leading edge following an epithelial abrasion, and this fine-tuned regulation is lost during the onset of diabetes. Further understanding of the corneal changes that occur in diabetes can help us better monitor progression of diabetic complications, as well as develop new therapeutics for the treatment of diabetic corneal dysfunction.

Identification of Anti-prion Compounds using a Novel Cellular Assay.

Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (Δ105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.

Purinoreceptor P2X7 Regulation of Ca(2+) Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury.

The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein-labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing.

Epithelial wounds induce differential phosphorylation changes in response to purinergic and EGF receptor activation.

Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleotides released after scratch wounding activate purinergic receptors, leading to a distinct phosphorylation profile on epidermal growth factor receptor (EGFR) compared with its natural ligand. ATP induced a 2- to 15-fold phosphorylation increase over control on EGFR Y974, Y1086, and Y1148, with minimal phosphorylation intensity on EGFR Y1173 compared with the level measured in response to epidermal growth factor. Differential phosphorylation induced by epidermal growth factor or ATP was site specific on Src, Shc, phospholipase Cγ, protein kinase C, focal adhesion kinase, paxillin, and mitogen-activated protein kinases 1, 12, and 13. After wounding, the P2Y2 receptor mRNA expression increased, and after knockdown, migration and Ca(2+) mobilization were impaired. To examine phosphorylation mediated by P2Y2, cells were cultured in media containing stable isotope-labeled amino acids, the receptor was knocked down, and the cells were stimulated. Mass spectrometry-based comparison of the phosphorylation profiles of control versus transfected cells revealed a 50-fold decrease in phosphorylation of EGFR Y974 and 1086, with no decrease in Y1173 phosphorylation. A similarfold decrease in Src Y421 and Y446 and paxillin Y118 was detected, indicating the far-reaching importance of the P2Y2 receptor in mediating migration.

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

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. [(3)H] cholesteryl ester accumulated in cells prelabeled with [(3)H] cholesterol, confirming that serum amyloid A mediated the movement of endogenous cholesterol. Cholesterol movement was dependent upon functional endolysosomes. The cholesterol oxidase-sensitive pool of cholesterol decreased in serum amyloid A-treated cells. Furthermore, the mechanism whereby serum amyloid A induced cholesterol trafficking was determined to be via activation of expression of secretory phospholipase A(2), group IIA (sPLA(2)) and sPLA(2)-dependent activation of sphingomyelinase. Interestingly, although neither tumor necrosis factor-α nor interferon-γ induced cholesterol trafficking, interleukin-1ß induced [(14)C] cholesteryl ester accumulation that was also dependent upon sPLA(2) and sphingomyelinase activities. Serum amyloid A activates smooth muscle cell interleukin-1ß expression, and although the interleukin-1-receptor antagonist inhibited the interleukin-1ß-induced cholesterol trafficking, it had no effect on the movement of cholesterol mediated by serum amyloid A. CONCLUSIONS: These data support a role for inflammation in endogenous smooth muscle cell cholesterol trafficking from the plasma membrane to the endoplasmic reticulum.

Aberrations in Cell Signaling Quantified in Diabetic Murine Globes after Injury.

The corneal epithelium is an avascular structure that has a unique wound healing mechanism, which allows for rapid wound closure without compromising vision. This wound healing mechanism is attenuated in diabetic patients, resulting in poor clinical outcomes and recurrent non-healing erosion. We investigated changes in cellular calcium signaling activity during the wound response in murine diabetic tissue using live cell imaging from both ex vivo and in vitro models. The calcium signaling propagation in diabetic cells was significantly decreased and displayed altered patterns compared to non-diabetic controls. Diabetic cells and tissue display distinct expression of the purinergic receptor, P2X7, which mediates the wound healing response. We speculate that alterations in P2X7 expression, interactions with other proteins, and calcium signaling activity significantly impact the wound healing response. This may explain aberrations in the diabetic wound response.

Age Dependent Changes in Corneal Epithelial Cell Signaling.

The cornea is exposed daily to a number of mechanical stresses including shear stress from tear film and blinking. Over time, these stressors can lead to changes in the extracellular matrix that alter corneal stiffness, cell-substrate structures, and the integrity of cell-cell junctions. We hypothesized that changes in tissue stiffness of the cornea with age may alter calcium signaling between cells after injury, and the downstream effects of this signaling on cellular motility and wound healing. Nanoindentation studies revealed that there were significant differences in the stiffness of the corneal epithelium and stroma between corneas of 9- and 27-week mice. These changes corresponded to differences in the timeline of wound healing and in cell signaling. Corneas from 9-week mice were fully healed within 24 h. However, the wounds on corneas from 27-week mice remained incompletely healed. Furthermore, in the 27-week cohort there was no detectable calcium signaling at the wound in either apical or basal corneal epithelial cells. This is in contrast to the young cohort, where there was elevated basal cell activity relative to background levels. Cell culture experiments were performed to assess the roles of P2Y2, P2X7, and pannexin-1 in cellular motility during wound healing. Inhibition of P2Y2, P2X7, or pannexin-1 all significantly reduce wound closure. However, the inhibitors all have different effects on the trajectories of individual migrating cells. Together, these findings suggest that there are several significant differences in the stiffness and signaling that underlie the decreased wound healing efficacy of the cornea in older mice.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder.

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Cell-cell interactions mediate the response of vascular smooth muscle cells to substrate stiffness.

The vessel wall experiences progressive stiffening with age and the development of cardiovascular disease, which alters the micromechanical environment experienced by resident vascular smooth muscle cells (VSMCs). In vitro studies have shown that VSMCs are sensitive to substrate stiffness, but the exact molecular mechanisms of their response to stiffness remains unknown. Studies have also shown that cell-cell interactions can affect mechanotransduction at the cell-substrate interface. Using flexible substrates, we show that the expression of proteins associated with cell-matrix adhesion and cytoskeletal tension is regulated by substrate stiffness, and that an increase in cell density selectively attenuates some of these effects. We also show that cell-cell interactions exert a strong effect on cell morphology in a substrate-stiffness dependent manner. Collectively, the data suggest that as VSMCs form cell-cell contacts, substrate stiffness becomes a less potent regulator of focal adhesion signaling. This study provides insight into the mechanisms by which VSMCs respond to the mechanical environment of the blood vessel wall, and point to cell-cell interactions as critical mediators of VSMC response to vascular injury.

Secretory phospholipase A2, group IIA is a novel serum amyloid A target gene: activation of smooth muscle cell expression by an interleukin-1 receptor-independent mechanism.

Atherosclerosis is a multifactorial vascular disease characterized by formation of inflammatory lesions. Elevated circulating acute phase proteins indicate disease risk. Serum amyloid A (SAA) is one such marker but its function remains unclear. To determine the role of SAA on aortic smooth muscle cell gene expression, a preliminary screen of a number of genes was performed and a strong up-regulation of expression of secretory phospholipase A(2), group IIA (sPLA(2)) was identified. The SAA-induced increase in sPLA(2) was validated by real time PCR, Western blot analysis, and enzyme activity assays. Demonstrating that SAA increased expression of sPLA(2) heteronuclear RNA and that inhibiting transcription eliminated the effect of SAA on sPLA(2) mRNA suggested that the increase was transcriptional. Transient transfections and electrophoretic mobility shift assays identified CAAT enhancer-binding protein (C/EBP) and nuclear factor kappaB (NFkappaB) as key regulatory sites mediating the induction of sPLA(2). Moreover, SAA activated the inhibitor of NF-kappaB kinase (IKK) in cultured smooth muscle cells. Previous reports showed that interleukin (IL)-1beta up-regulates Pla2g2a gene transcription via C/EBPbeta and NFkappaB. Interestingly, SAA activated smooth muscle cell IL-1beta mRNA expression, however, blocking IL-1 receptors had no effect on SAA-mediated activation of sPLA(2) expression. Thus, the observed changes in sPLA(2) expression were not secondary to SAA-induced IL-1 receptor activation. The association of SAA with high density lipoprotein abrogated the SAA-induced increase in sPLA(2) expression. These data suggest that during atherogenesis, SAA can amplify the involvement of smooth muscle cells in vascular inflammation and that this can lead to deposition of sPLA(2) and subsequent local changes in lipid homeostasis.

Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium.

Epithelial wound healing is essential to repair the corneal barrier function after injury and requires coordinated epithelial sheet movement over the wounded region. The presence and role of pannexin1 on multilayered epithelial sheet migration was examined in unwounded and wounded corneal epithelium from C57BL/6J (B6) control and diet-induced obese (DiO) mice, a pretype 2 diabetic model. We hypothesize that pannexin1 is dysregulated, and the interaction of two ion-channel proteins (P2X7 and pannexin1) is altered in pretype 2 diabetic tissue. Pannexin1 was found to be present along cell borders in unwounded tissue, and no significant difference was observed between DiO and B6 control. However, an epithelial debridement induced a striking difference in pannexin1 localization. The B6 control epithelium displayed intense staining near the leading edge, which is the region where calcium mobilization was detected, whereas the staining in the DiO corneal epithelium was diffuse and lacked distinct gradation in intensity back from the leading edge. Cells distal to the wound in the DiO tissue were irregular in shape, and the morphology was similar to that of epithelium inhibited with 10Panx, a pannexin1 inhibitor. Pannexin1 inhibition reduced mobilization of calcium between cells near the leading edge, and MATLAB scripts revealed a reduction in cell-cell communication that was also detected in cultured cells. Proximity ligation was performed to determine if P2X7 and pannexin1 interaction was a necessary component of motility and communication. While there was no significant difference in the interaction in unwounded DiO and B6 control corneal epithelium, there was significantly less interaction in the wounded DiO corneas both near the wound and back from the edge. The results demonstrate that pannexin1 contributes to the healing response, and P2X7 and pannexin1 coordination may be a required component of cell-cell communication and an underlying reason for the lack of pathologic tissue migration.

The Role of Hypoxia in Corneal Extracellular Matrix Deposition and Cell Motility.

The cornea is an excellent model tissue to study how cells adapt to periods of hypoxia as it is naturally exposed to diurnal fluxes in oxygen. It is avascular, transparent, and highly innervated. In certain pathologies, such as diabetes, limbal stem cell deficiency, or trauma, the cornea may be exposed to hypoxia for variable lengths of time. Due to its avascularity, the cornea requires atmospheric oxygen, and a reduction in oxygen availability can impair its physiology and function. We hypothesize that hypoxia alters membrane stiffness and the deposition of matrix proteins, leading to changes in cell migration, focal adhesion formation, and wound repair. Two systems-a 3D corneal organ culture model and polyacrylamide substrates of varying stiffness-were used to examine the response of corneal epithelium to normoxic and hypoxic environments. Exposure to hypoxia alters the deposition of the matrix proteins such as laminin and Type IV collagen. In addition, previous studies had shown a change in fibronectin after injury. Studies performed on matrix-coated acrylamide substrates ranging from 0.2 to 50 kPa revealed stiffness-dependent changes in cell morphology. The localization, number, and length of paxillin pY118- and vinculin pY1065-containing focal adhesions were different in wounded corneas and in human corneal epithelial cells incubated in hypoxic environments. Overall, these results demonstrate that low-oxygenated environments modify the composition of the extracellular matrix, basal lamina stiffness, and focal adhesion dynamics, leading to alterations in the function of the cornea. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

Sustained Ca2+ mobilizations: A quantitative approach to predict their importance in cell-cell communication and wound healing.

Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. We developed a method to characterize the injury-induced sustained Ca2+ mobilizations that travel between cells for periods of time up to several hours. These events of communication are concentrated along the wound edge and are reduced in cells further away from the wound. Our goal was to delineate the role and contribution of these sustained mobilizations and using MATLAB analyses, we determined the probability of cell-cell communication events in both in vitro models and ex vivo organ culture models. We demonstrated that the injury response was complex and represented the activation of a number of receptors. In addition, we found that pannexin channels mediated the cell-cell communication and motility. Furthermore, the sustained Ca2+ mobilizations are associated with changes in cell morphology and motility during wound healing. The results demonstrate that both purinoreceptors and pannexins regulate the sustained Ca2+ mobilization necessary for cell-cell communication in wound healing.

Inhibition of histone acetyltransferase by glycosaminoglycans.

Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans (GAGs) are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans (HSPGs) being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin-derived oligosaccharides (>8 sugars) and N-desulfated or O-desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive-like inhibitor causing an approximately 50-fold increase in the apparent Km of pCAF for histone H4. HSPGs isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous GAGs might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an approximately 50% reduction of histone H3 acetylation after 6 h of treatment. In addition, Chinese hamster ovary cells deficient in GAG synthesis showed increased levels of acetylated histone H3 compared to wild-type parent cells. GAGs represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation.

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. Although infection with periodontal and respiratory pathogens via activation of inflammatory cell Toll-like receptor (TLR)2 has been linked to vascular disease, little is known about smooth muscle cell TLR2 in atherosclerosis. This study addresses the role of SAA and TLR2 activation on smooth muscle cell matrix gene expression and insoluble elastin accumulation. Cultured rat aortic smooth muscle cells were treated with SAA or TLR2 agonists and the effect on expression of matrix metallopeptidase 9 (MMP9) and tropoelastin studied. SAA up-regulated MMP9 expression. Tropoelastin is an MMP9 substrate and decreased tropoelastin levels in SAA-treated cells supported the concept of extracellular matrix remodeling. Interestingly, SAA-induced down-regulation of tropoelastin was not only evident at the protein level but at the level of gene transcription as well. Contributions of proteasomes, nuclear factor κ B and CCAAT/enhancer binding protein ß on regulation of MMP9 vs. tropoleastin expression were revealed. Effects on Mmp9 and Eln mRNA expression persisted with long-term SAA treatment, resulting in decreased insoluble elastin accumulation. Interestingly, the SAA effects were TLR2-dependent and TLR2 activation by bacterial ligands also induced MMP9 expression and decreased tropoelastin expression. These data reveal a novel mechanism whereby SAA and/or infection induce changes in vascular elastin consistent with atherosclerosis.

The N-terminus of the prion protein is a toxic effector regulated by the C-terminus.

PrPC, the cellular isoform of the prion protein, serves to transduce the neurotoxic effects of PrPSc, the infectious isoform, but how this occurs is mysterious. Here, using a combination of electrophysiological, cellular, and biophysical techniques, we show that the flexible, N-terminal domain of PrPC functions as a powerful toxicity-transducing effector whose activity is tightly regulated in cis by the globular C-terminal domain. Ligands binding to the N-terminal domain abolish the spontaneous ionic currents associated with neurotoxic mutants of PrP, and the isolated N-terminal domain induces currents when expressed in the absence of the C-terminal domain. Anti-PrP antibodies targeting epitopes in the C-terminal domain induce currents, and cause degeneration of dendrites on murine hippocampal neurons, effects that entirely dependent on the effector function of the N-terminus. NMR experiments demonstrate intramolecular docking between N- and C-terminal domains of PrPC, revealing a novel auto-inhibitory mechanism that regulates the functional activity of PrPC.

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. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 microg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 microg CSC/ml. Moreover, 80-120 microg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema.