Distribution of Glycerophospholipids in the Adult Human Lens.
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ABSTRACT:
SUBMITTER: Seng JA
PROVIDER: S-EPMC6315977 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Knowledge of the spatial distribution of lipids in the intraocular lens is important for understanding the physiology and biochemistry of this unique tissue and for gaining a better insight into the mechanisms underlying diseases of the lens. Following our previous study showing the spatial distribution of sphingolipids in the porcine lens, the current study used ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) to provide the whole lipidome of porcine lens and these studies were supplemented by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) of the lens using ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to determine the spatial distribution of glycerophospholipids. Altogether 172 lipid species were identified with high confidence and their concentration was determined. Sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines were the most abundant lipid classes. We then determined the spatial and concentration-dependent distributions of 20 phosphatidylcholines, 6 phosphatidylethanolamines, and 4 phosphatidic acids. Based on the planar molecular images of the lipids, we report the organization of fiber cell membranes within the ocular lens and suggest roles for these lipids in normal and diseased lenses.
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Project description:The formation of an internal barrier to the diffusion of small molecules in the lens during middle age is hypothesized to be a key event in the development of age-related nuclear (ARN) cataract. Changes in membrane lipids with age may be responsible. In this study, we investigated the effect of age on the distribution of sphingomyelins, the most abundant lens phospholipids. Human lens sections were initially analyzed by MALDI mass spectrometry imaging. A distinct annular distribution of the dihydrosphingomyelin, DHSM (d18:0/16:0), in the barrier region was observed in 64- and 70-year-old lenses but not in a 23-year-old lens. An increase in the dihydroceramide, DHCer (d18:0/16:0), in the lens nucleus was also observed in the older lenses. These findings were supported by ESI mass spectrometry analysis of lipid extracts from lenses dissected into outer, barrier, and nuclear regions. A subsequent analysis of 18 lenses ages 20-72 years revealed that sphingomyelin levels increased with age in the barrier region until reaching a plateau at approximately 40 years of age. Such changes in lipid composition will have a significant impact on the physical properties of the fiber cell membranes and may be associated with the formation of a barrier.
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Project description:PurposeMetallothioneins (MTs) are a large family of proteins involved in multiple protective pathways including binding of toxic metals, free radical scavenging, and oxidative stress. Increased expression of the MT IIA gene in age-related cataractous relative to normal human lenses, suggesting a role for MTs in the maintenance of lens transparency, has previously been detected. The MT family consists of many closely related isoforms grouped into four classes (I-IV). As a first step toward defining the function of MTs in the lens, the range and expression patterns of those MT isoforms expressed by the adult human lens were established.MethodsNormal human lenses were microdissected into epithelia and fiber cells. Primers specific for individual MT isoforms were designed. MT transcripts were monitored in whole lenses, epithelia, and fibers by RT-PCR and confirmed to be authentic by sequencing. MT protein levels were evaluated by immunoblotting and by immunostaining.ResultsTranscripts encoding MT classes I and II but not III or IV were detected in adult human lenses. In addition to MT IIA, five other MT transcripts were identified including IE, IF, IG, IH, and IL. MT IIA was detected almost exclusively in the lens epithelium, whereas the class I isoforms were detected at high levels in both lens epithelia and fibers. MT protein was detected almost exclusively in the lens epithelium.ConclusionsThe present report establishes the spectrum of MTs expressed by the adult human lens, defines their spatial expression patterns in lens epithelia and fibers, and demonstrates that MT protein is abundantly present in the lens epithelium.
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Project description:Laminin N-terminus α31 (LaNt α31) is a netrin-like protein derived from alternative splicing of the laminin α3 gene. Although LaNt α31 has been demonstrated to influence corneal and skin epithelial cell function, its expression has not been investigated beyond these tissues. In this study, we used immunohistochemistry to characterise the distribution of this protein in a wide-array of human tissue sections in comparison to laminin α3. The data revealed widespread LaNt α31 expression. In epithelial tissue, LaNt α31 was present in the basal layer of the epidermis, throughout the epithelium of the digestive tract, and in much of the epithelium of the reproductive system. LaNt α31 was also found throughout the vasculature of most tissues, with enrichment in reticular-like fibres in the extracellular matrix surrounding large vessels. A similar matrix pattern was observed around the terminal ducts in the breast and around the alveolar epithelium in the lung, where basement membrane staining was also evident. Specific enrichment of LaNt α31 was identified in sub-populations of cells of the kidney, liver, pancreas, and spleen, with variations in intensity between different cell types in the collecting ducts and glomeruli of the kidney. Intriguingly, LaNt α31 immunoreactivity was also evident in neurons of the central nervous system, in the cerebellum, cerebral cortex, and spinal cord. Together these findings suggest that LaNt α31 may be functionally relevant in a wider range of tissue contexts than previously anticipated, and the data provides a valuable basis for investigation into this interesting protein.
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Project description:The content of polyunsaturated fatty acids (PUFA) in complex lipids essentially influences their physicochemical properties and has been linked to health and disease. To investigate the incorporation of dietary PUFA in the human plasma lipidome, we quantified glycerophospholipids (GPL), sphingolipids, and sterols using electrospray ionization coupled to tandem mass spectrometry of plasma samples obtained from a dietary intervention study. Healthy individuals received foods supplemented with different vegetable oils rich in PUFA. These included sunflower, linseed, echium, and microalgae oil as sources of linoleic acid (LA; FA 18:2 n-6), alpha-linolenic acid (ALA; FA 18:3 n-3), stearidonic acid (SDA; FA 18:4 n-3), and docosahexaenoic acid (DHA; FA 22:6 n-3). While LA and ALA did not influence the species profiles of GPL, sphingolipid, and cholesteryl ester drastically, SDA and DHA were integrated primarily in ethanolamine-containing GPL. This significantly altered phosphatidylethanolamine and plasmalogen species composition, especially those with 38-40 carbons and 6 double bonds. We speculate that diets enriched with highly unsaturated FA more efficiently alter plasma GPL acyl chain composition than those containing primarily di- and tri-unsaturated FA, most likely because of their more pronounced deviation of FA composition from typical western diets.
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Project description:Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.
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Project description:BackgroundThe limbus is located at a 2-mm-wide area between the bulbar conjunctiva and the cornea and has been suggested to be the niche of corneal epithelial stem cells and immune cells. Like the skin and intestines, the cornea is also an important mucosal surface, and immune cells on the cornea play critical roles in immune surveillance to ensure barrier surface homeostasis and protection from various environmental damage and infections. Single-cell RNA sequencing (scRNA-seq) analysis of protein tyrosine phosphatase receptor type C positive (PTPRC+) hematopoietic cells from the corneal limbus could provide a single cell atlas of all the immune cell subsets.MethodsWe performed single-cell RNA sequencing to generate transcriptomic profile for 804 sort-purified hematopoietic cells from the corneal limbus of three healthy donors.ResultsOur analysis identified a primary transcriptomic pattern for multiple immune cell subtypes, including naive T cells, antiviral effector CD8+ T cells, and innate immune cells such as IDO1+ mature regulatory dendritic cells (mregDCs), macrophages, monocytes, and basophils in the human corneal limbus.ConclusionOverall, single-cell transcriptomic analysis of limbal immune cells suggested the possible contribution of these cells on the adaptive and innate immune response of the human cornea.
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Project description:PurposePAX6 is a critical regulator of the developing lens, other ocular tissues, central nervous system, and pancreas. There are two alternatively spliced forms of the protein, PAX6 and PAX6(5a), that may have different regulatory functions. This study was designed to determine the amounts of PAX6 and PAX6(5a) transcripts present in adult human lens epithelium and fibers, human cornea and monkey retina.MethodsPAX6 and PAX6(5a) transcript levels were monitored in microdissected lens epithelia, lens fibers, whole lens, cornea, and retina by competitive RT-PCR. The levels of TBP/TFIID were examined in adult human lens epithelium and fibers as control.ResultsPAX6 and PAX6(5a) were expressed at equal levels in lens epithelium and fibers. Ninety-five times more PAX6 transcripts were detected in the epithelial cells than in the fibers. Adult human cornea and monkey retina expressed less PAX6/PAX6(5a) than lens epithelium but more than lens fibers. Correspondingly, 40 fold higher levels of TBP transcripts were detected in lens epithelium than fibers, suggesting reduced overall expression of transcription factors in the adult lens fibers.ConclusionsThe presence of PAX6 and PAX6(5a) messages and proteins in adult lens epithelium suggest functions for both forms of PAX6 in the growth and maintenance of the adult human lens. The reduced levels of both forms of PAX6 in the lens fibers suggest down regulation of this gene during differentiation of epithelia into fibers. The lower level of TBP expression in lens fibers also suggests reduced transcriptional competence of adult lens fibers.
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