This study utilizes high resolution multi-dimensional imaging to identify temporal and spatial changes in cell/extracellular matrix (ECM) patterning mediating cell migration, fibrosis, remodeling and regeneration during wound healing. Photorefractive keratectomy (PRK) was performed on rabbits. In some cases, 5([4,6-dichlorotriazin-2yl]-amino)fluorescein (DTAF) was applied immediately after surgery to differentiate native vs. cell-secreted collagen. Corneas were assessed 3–180 days postoperatively using in vivo confocal microscopy, and cell/ECM patterning was evaluated in situ using multiphoton and second harmonic generation (SHG) imaging. 7 days post-PRK, migrating fibroblasts below the ablation site were co-aligned with the stromal lamellae. At day 21, randomly patterned myofibroblasts developed on top of the ablation site; whereas cells underneath were elongated, co-aligned with collagen, and lacked stress fibers. Over time, fibrotic tissue was remodeled into more transparent stromal lamellae. By day 180, stromal thickness was almost completely restored. Stromal regrowth occurred primarily below the ablation interface, and was characterized by co-localization of gaps in DTAF labeling with elongated cells and SHG collagen signaling. Punctate F-actin labeling was detected along cells co-aligned with DTAF and non-DTAF labeled collagen, suggesting cell-ECM interactions. Overall, collagen lamellae appear to provide a template for fibroblast patterning during wound healing that mediates stromal repopulation, regeneration and remodeling.
Introduction: HMG-CoA reductase is a membrane protein of the endoplasmic reticulum that catalyzes reduction of HMG-CoA to mevalonate, a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, which exert feedback control on HMGCR through multiple mechanisms. These mechanisms ensure constant synthesis of essential nonsterol isoprenoids, while avoiding toxic cholesterol accumulation. One mechanism involves sterol-induced ubiquitination of HMGCR, marking the enzyme for degradation from ER membranes, a process augmented by nonsterol isoprenoids. We examine the contribution of sterol-accelerated ubiquitination/degradation to overall regulation of HMGCR in livers of mice. Methods: Forty mice, including 20 wild-type (WT) and 20 knock-in (Ki) mice expressing ubiquitination-resistant HMGCR, were fed diets containing only chow, or chow supplemented with 0.1, 0.3, or 1% cholesterol. After five days of feeding, livers were harvested for measurements of cholesterol and triglycerides, immunoblot analysis, and qRT-PCR of genes related to cholesterol, nonsterol isoprenoid, and fatty acid synthesis. Results: Normalization of mRNA to protein levels indicates that HMGCR Ki livers contain a more HMGCR protein despite mRNA downregulation. Protein and gene expression of SREBP2 and its target genes, which contribute to cholesterol synthesis, decreased as expected with increased dietary cholesterol. Conversely, protein and gene expression of SREBP1 and its target genes increased, likely due to SREBP1c predominance toward fatty acid synthesis, which prevents cholesterol accumulation. Conclusion: The increase in HMGCR protein relative to mRNA suggests that significant post-transcriptional regulation exists in the form of impaired degradation. Furthermore, these normalized values indicate that accumulation of protein is primarily due to impaired degradation at lower cholesterol levels (chow, 0.1%); however, at high cholesterol levels (0.3, 1%), a greater degree of transcriptional control from sterol-mediated inhibition of SREBP2 regulates HMGCR due to negative feedback. This study demonstrates the role of degradative control on inhibition of HMGCR and may assist in reducing HMGCR accumulation during statin therapy.
IntroductionHMG‐CoA reductase is a membrane protein of the endoplasmic reticulum (ER) that catalyzes the reduction of HMG‐CoA to mevalonate, a rate‐limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Sterol and nonsterol isoprenoids exert stringent feedback control on HMGCR through multiple mechanisms. This ensures constant synthesis of essential nonsterol isoprenoids, while avoiding toxic overaccumulation of cholesterol. One of these mechanisms involves sterol‐induced ubiquitination of HMGCR, which marks the enzyme for degradation from ER membranes that is augmented by nonsterol isoprenoids. In this study, we examine the contribution of this sterol‐accelerated ubiquitination/degradation to overall regulation of HMGCR in the livers of mice.MethodsForty mice, including 20 wild‐type (WT) and 20 knock‐in (Ki) mice that express ubiquitination‐resistant HMGCR, were fed diets containing only chow, or chow supplemented with 0.1%, 0.3%, or 1% cholesterol. After five days of feeding, livers were harvested for measurements of cholesterol and triglycerides, immunoblot analysis of six proteins, and qRTPCR of 26 genes related to cholesterol, nonsterol isoprenoid, and fatty acid synthesis.ResultsNormalization of mRNA levels to protein levels indicates that HMGCR Ki mouse livers contain a higher level of HMGCR protein despite mRNA downregulation. Protein and gene expression of SREBP‐2 and its target genes, which contribute to cholesterol synthesis, decreased as expected with increased dietary cholesterol. Conversely, protein and gene expression of SREBP‐1 and its target genes increased, likely due to SREBP‐1c predominance toward fatty acid synthesis, which prevents cholesterol accumulation.ConclusionThe increase in HMGCR protein relative to mRNA suggests that significant posttranscriptional regulation exists in the form of impaired degradation. Furthermore, these normalized values indicate that accumulation of protein is primarily due to impaired degradation at lower cholesterol levels (chow, 0.1%); however, at high cholesterol levels (0.3%, 1%), a greater degree of transcriptional control from sterol‐mediated inhibition of SREBP‐2 regulates HMGCR due to negative feedback. This study demonstrates the role of degradative control on inhibition of HMGCR and may assist in reducing HMGCR accumulation during statin therapy.Support or Funding InformationNIH grants HL020948, GM112409, Ruth L. Kirschstein National Research Service Award (NRSA) Short‐Term Institutional Research Training Grant (T‐35)
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