Celeste Rich - Academia.edu (original) (raw)

Papers by Celeste Rich

Journal of Biological Chemistry, 1978

The P2X 7 receptor regulates proteoglycan expression in the corneal

Journal of Functional Biomaterials, 2011

The American Journal of Pathology, 2011

The release of nucleotides after injury activates purinergic receptors, leading to phosphorylatio... more The release of nucleotides after injury activates purinergic receptors, leading to phosphorylation of site-specific residues on epidermal growth factor receptor (EGFR). To elucidate the differences between the injuryinduced response and that induced by exogenous EGF, we examined recruitment of docking proteins, internalization of EGFR, and migration after injury. Injury induced by scratch wounds or stimulation by addition of UTP caused a brief internalization of EGFR, which paralleled the lesser association with growth factor receptor-bound protein 2 (Grb2) and phosphorylation of EGFR. The internalization caused by EGF was sustained and detected for longer than 60 minutes and correlated with phosphorylation of the receptor. The EGF caused recruitment of Grb2, phospholipase C-␥-1 (PLC␥1), Shc, and Src to EGFR. Glutathione S-transferase pull downs were performed, and glutathione S-transferase-PLC␥1 showed binding of Grb2 when stimulated with EGF but not with UTP or injury. Furthermore, UTP did not induce PLC␥1 phosphorylation, and the phosphorylation induced by EGF was attenuated by costimulation with UTP. The response to heparin-binding EGF was equivalent to that of EGF. Site-directed mutagenesis showed that phosphorylation of Y1068 and Y1086 of EGFR is required for repair. Together, our results show that injury and activation of purinergic receptors and direct activation of EGFR via EGF induce distinct downstream pathways.

C27. GENETIC PROGRAMS DRIVING LUNG DEVELOPMENT AND REGENERATION, 2011

Journal of Cellular Biochemistry, 2008

Journal of Biological Chemistry, 2009

Biophysical Journal, 2011

The vessel wall experiences progressive stiffening with age and the development of cardiovascular... more 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.

Biophysical Journal, 2009

Arteriosclerosis, Thrombosis, and Vascular Biology, 2012

Objective-Intracellular cholesterol distribution impacts cell function; however, processes influe... more 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-1β induced [ 14 C] cholesteryl ester accumulation that was also dependent upon sPLA 2 and sphingomyelinase activities. Serum amyloid A activates smooth muscle cell interleukin-1β expression, and although the interleukin-1receptor antagonist inhibited the interleukin-1β−induced cholesterol trafficking, it had no effect on the movement of cholesterol mediated by serum amyloid A. Conclusion-These data support a role for inflammation in endogenous smooth muscle cell cholesterol trafficking from the plasma membrane to the endoplasmic reticulum.

American Journal of Physiology-Lung Cellular and Molecular Physiology, 2006

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated wit... more 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). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-α-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-β signaling, because TGF-β type I receptor kinase inhibitor or TGF-β neutralizing antibody ...

Investigative Opthalmology & Visual Science, 2008

PURPOSE. Previously, the authors demonstrated that BzATP, a P2X 7 receptor agonist, enhanced corn... more PURPOSE. Previously, the authors demonstrated that BzATP, a P2X 7 receptor agonist, enhanced corneal epithelial migration in vitro. The goal here was to characterize the role of the P2X 7 receptor in the repair of in vivo corneal epithelial debridement wounds and in the structural organization of the corneal stroma. METHODS. Epithelial debridement was performed on P2X 7 knockout (P2X 7 Ϫ/Ϫ) and wild-type (WT) mice, and eyes were harvested after 16 hours. Corneas were stained with Richardson vital stain, and the wound area was recorded. Corneas were fixed and prepared for light microscopic, immunohistochemical, and electron microscopic analysis. Cuprolinic blue staining was performed to analyze stromal proteoglycans (PGs). Real-time PCR was performed to examine the expression of stromal collagens. RESULTS. P2X 7 was present in the WT corneal epithelium but was not detected in P2X 7 Ϫ/Ϫ mice. Pannexin-1, a protein demonstrated to interact with P2X 7 , was absent from the wound edge in P2X 7 Ϫ/Ϫ. This was associated with a trend toward delayed corneal reepithelialization. Stromal ultrastructure and collagen alignment were altered in P2X 7 Ϫ/Ϫ , and collagen fibrils had smaller diameters with a larger interfibrillar distances. Expression of collagen alpha1(I) and alpha3(v) was reduced. There were 30% fewer sulfated PGs along fibrils in the P2X 7 Ϫ/Ϫ stroma. CONCLUSIONS. In the absence of the P2X 7 receptor, the expression of proteins in the corneal epithelium was altered and wound healing was compromised. Loss of receptor resulted in morphologic changes in the stroma, including changes in alignment of collagen fibrils, decreased expression of collagen, and smaller fibrils with fewer PGs per fibril. (Invest Ophthalmol

Investigative Opthalmology & Visual Science, 2008

Bioengineering

The cornea is avascular, which makes it an excellent model to study matrix protein expression and... more The cornea is avascular, which makes it an excellent model to study matrix protein expression and tissue stiffness. The corneal epithelium adheres to the basement zone and the underlying stroma is composed of keratocytes and an extensive matrix of collagen and proteoglycans. Our goal was to examine changes in corneas of 8- and 15-week mice and compare them to 15-week pre-Type 2 diabetic obese mouse. Nanoindentation was performed on corneal epithelium in situ and then the epithelium was abraded, and the procedure repeated on the basement membrane and stroma. Confocal imaging was performed to examine the localization of proteins. Stiffness was found to be age and obesity dependent. Young’s modulus was greater in the epithelium from 15-week mice compared to 8-week mice. At 15 weeks, the epithelium of the control was significantly greater than that of the obese mice. There was a difference in the localization of Crb3 and PKCζ in the apical epithelium and a lack of lamellipodial extensio...

International Journal of Molecular Sciences

Vascular endothelial growth factor-A (VEGF) is critical for the development, growth, and survival... more 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...

Journal of Biological Chemistry, 1978

The P2X 7 receptor regulates proteoglycan expression in the corneal

Journal of Functional Biomaterials, 2011

The American Journal of Pathology, 2011

The release of nucleotides after injury activates purinergic receptors, leading to phosphorylatio... more The release of nucleotides after injury activates purinergic receptors, leading to phosphorylation of site-specific residues on epidermal growth factor receptor (EGFR). To elucidate the differences between the injuryinduced response and that induced by exogenous EGF, we examined recruitment of docking proteins, internalization of EGFR, and migration after injury. Injury induced by scratch wounds or stimulation by addition of UTP caused a brief internalization of EGFR, which paralleled the lesser association with growth factor receptor-bound protein 2 (Grb2) and phosphorylation of EGFR. The internalization caused by EGF was sustained and detected for longer than 60 minutes and correlated with phosphorylation of the receptor. The EGF caused recruitment of Grb2, phospholipase C-␥-1 (PLC␥1), Shc, and Src to EGFR. Glutathione S-transferase pull downs were performed, and glutathione S-transferase-PLC␥1 showed binding of Grb2 when stimulated with EGF but not with UTP or injury. Furthermore, UTP did not induce PLC␥1 phosphorylation, and the phosphorylation induced by EGF was attenuated by costimulation with UTP. The response to heparin-binding EGF was equivalent to that of EGF. Site-directed mutagenesis showed that phosphorylation of Y1068 and Y1086 of EGFR is required for repair. Together, our results show that injury and activation of purinergic receptors and direct activation of EGFR via EGF induce distinct downstream pathways.

C27. GENETIC PROGRAMS DRIVING LUNG DEVELOPMENT AND REGENERATION, 2011

Journal of Cellular Biochemistry, 2008

Journal of Biological Chemistry, 2009

Biophysical Journal, 2011

The vessel wall experiences progressive stiffening with age and the development of cardiovascular... more 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.

Biophysical Journal, 2009

Arteriosclerosis, Thrombosis, and Vascular Biology, 2012

Objective-Intracellular cholesterol distribution impacts cell function; however, processes influe... more 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-1β induced [ 14 C] cholesteryl ester accumulation that was also dependent upon sPLA 2 and sphingomyelinase activities. Serum amyloid A activates smooth muscle cell interleukin-1β expression, and although the interleukin-1receptor antagonist inhibited the interleukin-1β−induced cholesterol trafficking, it had no effect on the movement of cholesterol mediated by serum amyloid A. Conclusion-These data support a role for inflammation in endogenous smooth muscle cell cholesterol trafficking from the plasma membrane to the endoplasmic reticulum.

American Journal of Physiology-Lung Cellular and Molecular Physiology, 2006

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated wit... more 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). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-α-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-β signaling, because TGF-β type I receptor kinase inhibitor or TGF-β neutralizing antibody ...

Investigative Opthalmology & Visual Science, 2008

PURPOSE. Previously, the authors demonstrated that BzATP, a P2X 7 receptor agonist, enhanced corn... more PURPOSE. Previously, the authors demonstrated that BzATP, a P2X 7 receptor agonist, enhanced corneal epithelial migration in vitro. The goal here was to characterize the role of the P2X 7 receptor in the repair of in vivo corneal epithelial debridement wounds and in the structural organization of the corneal stroma. METHODS. Epithelial debridement was performed on P2X 7 knockout (P2X 7 Ϫ/Ϫ) and wild-type (WT) mice, and eyes were harvested after 16 hours. Corneas were stained with Richardson vital stain, and the wound area was recorded. Corneas were fixed and prepared for light microscopic, immunohistochemical, and electron microscopic analysis. Cuprolinic blue staining was performed to analyze stromal proteoglycans (PGs). Real-time PCR was performed to examine the expression of stromal collagens. RESULTS. P2X 7 was present in the WT corneal epithelium but was not detected in P2X 7 Ϫ/Ϫ mice. Pannexin-1, a protein demonstrated to interact with P2X 7 , was absent from the wound edge in P2X 7 Ϫ/Ϫ. This was associated with a trend toward delayed corneal reepithelialization. Stromal ultrastructure and collagen alignment were altered in P2X 7 Ϫ/Ϫ , and collagen fibrils had smaller diameters with a larger interfibrillar distances. Expression of collagen alpha1(I) and alpha3(v) was reduced. There were 30% fewer sulfated PGs along fibrils in the P2X 7 Ϫ/Ϫ stroma. CONCLUSIONS. In the absence of the P2X 7 receptor, the expression of proteins in the corneal epithelium was altered and wound healing was compromised. Loss of receptor resulted in morphologic changes in the stroma, including changes in alignment of collagen fibrils, decreased expression of collagen, and smaller fibrils with fewer PGs per fibril. (Invest Ophthalmol

Investigative Opthalmology & Visual Science, 2008

Bioengineering

The cornea is avascular, which makes it an excellent model to study matrix protein expression and... more The cornea is avascular, which makes it an excellent model to study matrix protein expression and tissue stiffness. The corneal epithelium adheres to the basement zone and the underlying stroma is composed of keratocytes and an extensive matrix of collagen and proteoglycans. Our goal was to examine changes in corneas of 8- and 15-week mice and compare them to 15-week pre-Type 2 diabetic obese mouse. Nanoindentation was performed on corneal epithelium in situ and then the epithelium was abraded, and the procedure repeated on the basement membrane and stroma. Confocal imaging was performed to examine the localization of proteins. Stiffness was found to be age and obesity dependent. Young’s modulus was greater in the epithelium from 15-week mice compared to 8-week mice. At 15 weeks, the epithelium of the control was significantly greater than that of the obese mice. There was a difference in the localization of Crb3 and PKCζ in the apical epithelium and a lack of lamellipodial extensio...

International Journal of Molecular Sciences

Vascular endothelial growth factor-A (VEGF) is critical for the development, growth, and survival... more 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...