Corneal Stroma Lecture (PDF)

Corneal Stroma Roy Joseph PhD, MBA School of Optometry and Vision Science University of Alabama at Birmingham 09/03/2024 Ocular Anatomy and Biology (VIS 610)  Layers of Cornea C...

Corneal Stroma Roy Joseph PhD, MBA School of Optometry and Vision Science University of Alabama at Birmingham 09/03/2024 Ocular Anatomy and Biology (VIS 610)  Layers of Cornea Corneal epithelium Bowman’s 50 m,10% of total layer, 8-12 m Stroma, 500 m, Collagen 90% of total Descemet’s membrane, 10 m Endothelium, 5 m  Collagen Secreted by connective tissue cells. >25 different - Collagen fibrils are made of three chains stranded polypeptide units called Many with all three ‘tropocollagen’ chains with Mr 300,000, 100 amino acids and identical amino 300 nm long. acids or two chains Arranged head to tail as parallel are identical, and bundles showing ‘cross-striations’ third chain is Amino acids: 35% Gly, 11% different Ala and 21% Pro Type I: 1(I)22(I). Rich in Pro (Ring structure stabilizes helical conformation of each chain). Gly as every third residue (small structure, so three chains pack Collagen Biosynthesis Comparison of Amino acid composition in Fibrous and Globular Proteins Fibrous Collagen and Elastin Globular Ribonuclease Hemoglobin Triple Helix Model of Collagen Gl y Gl y Gly  Classification of Collagen Types Most common in cornea, 58% 15 % Transmission electron microscopy of the developing corneal stroma at E14 showed high resolution images of the collagen fibrils. Transmission electron microscopy of the developing corneal stroma (A,B) At lower magnification, the collagen fibrils were seen to be abundant, surrounding the cells of the corneal stroma. (A) The collagen fibrils appeared to have started to cluster within bundles, with the bundles arranged orthogonally between cells. (A) An abundance of cell projections was seen within the corneal stroma, with some associating with the collagen fibrils (A, red arrows). The collagen fibrils were imaged at a higher magnification to show the orthogonal arrangement more clearly (B). Feneck, E.M., Lewis, P.N. & Meek, K.M. Three-dimensional imaging of the extracellular matrix and cell interactions in the developing prenatal mouse cornea. Sci Rep 9, 11277 (2019). Transmission electron microscopy imaging of the developing corneal stromal cells at E16 (A) and E18 (B) Transmission electron microscopy imaging of the developing corneal stromal cells at E16 (A) and E18 (B). With maturation, the amount of collagen fibrils was seen to increase until organized orthogonal lamellae were present. There was a tendency for cell projections (black arrow) within the corneal stroma of the developing corneas at E16 and E18 to align in the same direction as some of the immediately adjacent collagen fibrils. Feneck, E.M., Lewis, P.N. & Meek, K.M. Three-dimensional imaging of the extracellular matrix and cell interactions in the developing prenatal mouse cornea. Sci Rep 9, 11277 (2019). Structure of Collagen Fibrils  Cellular Interaction of Collagen Lamellae J Biol Chem. 2008 Jul 25;283(30):21187-97. doi: 10.1074/jbc.M709319200. Epub 2008 May 15.  Stromal Collagen Collagen: A structural Keratocytes: Most common protein organized into Secrete collagen collagen in cornea relatively inextensible and the is type I (58%), scaffold of water polypeptides of which aggregate insoluble fibrils that collagen into stranded form the basic molecules are banded fibrils. structural framework cleaved, and their of a connective tissue. Type I collagen is monomers are Stroma collagen: assembled into: generally Fibril forming or heterotype (type I Functionally important fibril- associated and type V [15%] to: collagen with collagen (A) Establish tissue’s interrupted molecules). This transparency (B) terminals (FACIT) may serve as fine Resisting tensile loads in a surface tuning mechanism and ultimately defining recess on the for controlling a size and shape of the keratocytes or fibril’s structure tissue. Of 28 known collagen, sometimes characteristics, begins assembly such as fibril size 13 are found in human inside the cells. and interfibrillar  Type I Collagen in Corneal Stroma -Type I Collagen reach certain -The fibrils typically of 25 nm diameter in the stroma specific diameter based on their with slight variability. composition and ratio of -The diameter of type I collagen fibrils remains heterotypic collagen molecule constant across most of the central cornea (mean: type and is restricted from further 25±2nm; range 18-32 nm) but gradually thickens lateral acceleration or growth. another 4 nm at about 5.5 nm from the center of the -The fibrils are permitted to fuse cornea and eventually increasing to 50 nm in limbus). and grow axially due to -The interfibrillar spacing between nearest neighbor interaction with small leucine-rich type I fibrils remains constant in central cornea proteoglycans (covalently bound (mean: 20 ±5 nm; range 5-35 nm) and then gradually to external surface of fibrils) and increases another 5 nm at about 4.5 to 5 nm from through surface interactions and center of the cornea). Type I fibril diameter and connect to various other fibril interfibrillar spacing do not seem to differ with depth forming, non-fibril forming or of the cornea. FACIT collagens. -Refractive index of type I fibril (1.47) is different from -The surface properties of type I extracellular matrix (1.35). fibrils are a major determinant of -The highly uniform small size fibrils, and highly interfibrillar and intrafibrillar uniform interfibrillar spaces along parallel biomechanical properties of the directionality result in lattice like structure. This short tissue. range arrangement allows corneal transparency by destructive interference. Collagen Fibril  Type V (15%) Type I (58%) Typ Type V eI Type VI Aging Figure 4.6 (A,B) Cross-sectional oblique view of a 25 nm diameter, heterotypic, banded (periodicity = 65 nm) corneal stromal collagen fibril composed of type I (white) and V (blue) collagen molecules (bottom). The amino-terminal domains on the type V collagen molecules appear to be important in regulating collagen fibril diameter as they project externally to the fibril surface and presumably block further accretion of collagen molecules through steric and/or electrostatic hindrance effects. The collagen molecules on longitudinal view are aligned in a parallel, quarter-staggered (68 nm) arrangement with 40 nm gaps between molecules (middle). The longitudinal view also clearly shows that the ends of the alpha chains in each collagen molecule form intermolecular cross-links with adjacent collagen molecules as well as intramolecular cross-links (top). (C) With maturity, these immature divalent cross-links become mature trivalent cross-links with the addition of interfibrillar cross-link branches. Finally, with aging, intramolecular, intermolecular, and interfibrillar non- enzymatic glycation cross-links form.  The collagen molecules on longitudinal view are aligned in a parallel, quarter- staggered (68 nm) arrangement with 40 Type V nm gaps between molecules (middle). The longitudinal view also clearly shows that the ends of the alpha chains in each collagen molecule form intermolecular Type I cross-links with adjacent collagen molecules as well as intramolecular cross-links (top). Intermolecular cross-links Diameter =25 nm, Periodicity = 65 nm The amino-terminal domains on the type V collagen molecules appear to be important in regulating collagen fibril diameter as they project externally to the fibril surface and presumably block further accretion of collagen molecules through steric and/or electrostatic hindrance effects.  Figure 4.7 (A) Low-magnification (×4750) TEM of predominantly orthogonally stacked lamellae in the middle third of stroma proper. Orthogonally (B) Higher magnification (×72,500) TEM stacked of two lamellae in the middle third of lamellae stroma proper. One lamella is in longitudinal view (top portion) and the other is in cross-sectional view (bottom portion). Longitudinal Notice the uniform 25 nm diameter type view (A lamellae) I collagen fibrils and uniform 20 nm diameter interfibrillar spaces, which Cross-sectional demonstrates only a short-range order, view but not a true crystalline lattice arrangement. Size of a wavelength (C) Cross-sectional diagram of collagen of light fibrils arranged in a true crystalline lattice arrangement. Size of a Cross-sectional wavelength of light is shown above for diagram of collagen comparison. fibrils arranged in (Modified from Maurice DM. J Physiol a true crystalline 1957; 136(2):263–86). lattice arrangement  Keratocytes Second major In adulthood, Immunohistochemical and component of stromal keratocytes occupy electron microscopic dry weight. 10% of stromal studies suggest that not They are sandwiched volume decreasing all cells are keratocytes, between collagenous 20% from in infancy. but three types of bone- lamella forming a Anterior 1/3 stroma marrow derived immune closed highly organized has higher keratocyte cells: syncytium. density and 2X more 1. professional dendritic During neonatal life, number of cells, keratocytes function as mitochondria in 2.non-professional fibroblasts forming keratocytes than dendritic cells and extracellular matrix of posterior in 2/3 of 3.histiocytes. stroma. Later, they exist stroma. Immune cells appear to as modified fibrocytes Keratocytes are play a pivotal role in the and maintain highly spatially induction of immune extracellular matrix. ordered as they turn tolerance vs. cell- If stroma is wounded, in a clock-wise mediated immunity and they become fibroblasts direction like cork stromal histiocytes act as screw. phagocytic effector cells.  Transmission Electron Microscopic Images Figure 4.11 Light and TEM photomicrographs showing in cross-section and tangential-section the keratocytes in the stroma. (A) Cross-sectional light microscopy view shows that keratocytes are primarily obliquely aligned to corneal surface in the anterior-third of cellular corneal stroma or are aligned parallel to the corneal surface in the posterior two-thirds. (B) Cross-sectional TEM view shows that keratocyte nuclei occupy most of the area of the keratocyte seen in this perspective with only a thin rim of surrounding cytoplasm that contains only small numbers of cytoplasmic organelles. (C) Tangential-section light microscopy view shows that keratocytes are arranged in a circular fashion. (D) Tangential-section TEM view shows that the supposedly quiescent keratocytes may actually be more active in the base-line state than initially thought as an extensive amount of cytoplasmic organelles can be seen in this view. M = mitochondria, RER = rough endoplasmic reticulum, V = vacuoles, * = main portion of nucleus that contains nucleolus. Keratocytes in the stroma (Modified from Muller LJ et al. Invest Ophthalmol Vis Sci Anterior third keratocytes are obliquely aligned 1995; 36:2557–67.) Posterior two thirds aligned parallel  Corneal Stromal Cell Types Immature dendritic cells Epitheli Mature um dendritic Corneal cells stromal cells Macropha Dendritic cells are ge involved in autoimmune response Figure 4.13 The mouse cornea suggests that 5–10 percent of the cells in the cellular corneal stroma are actually one of three types of bone marrow-derived immune cells. The anterior one third of the cellular corneal stroma contains resident Bone marrow derived immature Dendritic cells in the center and more mature dendritic cells in the periphery The. Posterior-most regions of the cellular corneal stroma appear to contain macrophages in the periphery and center of the cornea. (Modified from Hamrah P et al. Invest Ophthalmol Vis Sci 2003; 44:581–9.) Figure 4.12 (A) Reconstruction of keratocyte outlines seen in tangential- section in the anterior and posterior third of the corneal stroma proper. (Modified from Muller LJ et al. Invest Ophthalmol Vis Sci 1995; 36:2557–67.) Fluorescent dye spreading between many adjacent keratocytes in rabbit (center-left) and human corneas (center-right), which demonstrates the intimate importance of gap junctions in communication of keratocytes with one another. (From Watsky MA. Invest Ophthalmol Vis Sci 1995; 36:S22.)  Keratocyte Density Highest density The clinical haze seen in the early months after photorefractive keratectomy (PRK) may in part be due to an increased density of keratocytes. Bowma n’s layer (C) Mean stromal cell density associated with depth in the corneal stroma is shown. The highest zone of increased cell density was closest to the Bowman's layer. Perhaps this is due to baseline, normal epithelial–stromal interactions. (From Patel SV et al. Invest Ophthalmol Vis Sci 2001; 42:333–9.)  Corneal Stromal Proteoglycan Proteoglycans are macromolecules composed of a protein core with covalently linked glycosaminoglycan side chains. Decorin: Contains single dermatan sulfate GAG side chain ( 10 leucine repeat) Lumican and Mimican: Contain single keratan sulfate GAG side chain. Keratocan: Contains three keratan sulfate GAG side chains (small leucine rich repeats, (SLRP) So, four known types of proteoglycan core proteins and two types of GAGS, keratan sulfate (60%) and dermatan sulfate (40%) in stroma Keratan sulfate: Polymer of repeating units of galactose and N-acetyl glucosamine Dermatan sulfate: Polymer of repeating units of iduronic acid and N- acetylgalactosamine Glycosaminoglycan's  Keratan Sulfate Polymer of repeating units of galactose and N-acetyl glucosamine galactose N-acetyl glucosamine Like other glycosaminoglycans keratan sulfate is a linear polymer that consists of a repeating disaccharide unit. Keratan sulfate occurs as a proteoglycan (PG) in which KS chains are attached to cell-surface or extracellular matrix proteins, termed core proteins. KS core proteins include Lumican, Keratocan, Mimecan, Fibromodulin, PRELP, Osteoadherin and Aggrecan.  Dermatan Sulfate Polymer of repeating units of iduronic acid and N-acetylgalactosamine Iduronic acid N-acetylgalactosamine  Corneal Stromal Proteoglycans -Third major component of stroma Primary Using a electron-dense cationic dye called cupromeronic blue and 0.1 MgCl (that stains 2, -Water soluble glycoproteins made function: sulfate ester group) showed: Proteoglycans are not of core proteins with 1. Provide amorphous but tadpole shaped molecules made of covalently attached anionic tissue 10-15 nm diameter globular core protein with a polysaccharide side chain called volume, covalently attached 7 nm wide X 45-70 nm length glycosamine glycan (GAG). maintain (GAG side chain attach to the core proteins) spatial order -Arranged in corneal stroma perpendicular to -The core protein is non-covalently of collagen collagen fibrils with a constant spacing of 65 nm attached to collagen fibrils fibrils, resist between each other along with collagen fibrils. uniformly throughout the tissue. compression -The core proteins non-covalently bind to collagen forces, and fibrils in specific gap zones along the peripheral -The GAG side-chains extends into provide portions of the collagen fibrils. the interfibrillar space where it viscoelastic -Core protein with dermatan sulfate side-chains acts as a pressure-exerting property to binds to ‘d’ and ‘e’gap zones and those with polyelectrolyte gel. tissue. keratan sulfate side chains bind to ‘a’ and ‘c’ gap 2. Secondary zones -Corneas collapses 20% if role of -GAGs are highly negatively charged-stiff polymers proteoglycan is precipitated out regulating that extend into interfibrillar space and form anti- with cetylpyridinium. collagen fibril parallel duplexes with adjacent GAG side chains assembly. thereby linking nearest neighbor collagen fibrils by forming dumb-bell shaped structure.  With Without Cupromeronic Cupromeronic blue blue (stains Sulfate ester group) Type 1 corneal stromal collagen in Secondary lamella structures of each proteoglycan Figure 4.14 Tangential-section TEM views (×90,000) of longitudinally running type I corneal stromal collagen fibrils in a lamellae without (A) and with (B) cupromeronic blue staining. Notice the scattered “amorphous ground substance” in the interfibrillar spaces in (A), while in (B) duplexes of proteoglycans are clearly seen bridging next-nearest neighbor collagen fibrils. (C) Diagram of how proteoglycans attach along the periphery of type I fibrils via their core proteins (P) and how the core protein tail/GAGs duplex in an anti-parallel fashion in the interfibrillar space between next-nearest neighbor collagen fibrils. (D) Diagram of the polymer backbones of keratan sulfate and dermatan sulfate. The top portion shows the primary structure of the repeating disaccharide units of keratan sulfate (top left) and dermatan sulfate (top right). The bottom portion of the diagram shows the secondary structures of each proteoglycan. In the human cornea, ∼50 percent of keratan sulfate is in the normal-sulfated state (left), while the other ∼50 percent i in the over-sulfated state (center). All three displayed proteoglycan polymers have similar backbones and, therefore, form similar secondary structures of a two-fold helix (right). Anionic charges = sulfate esters (X) and carboxylic acid (−). (C and D are modified from Scott JE. Biochem Soc Trans 1991; 19:877–81.)  Collagen fibrils Core protein Core protein tail/GAGs duplex in an anti-parallel fashion in the interfibrillar space between next-nearest neighbor collagen fibrils.  Collagen fibrils Core protein tail/GAGs duplex in an anti-parallel fashion in the interfibrillar space between next-nearest neighbor collagen fibrils. Cuprolinic blue positively stained the glycosaminoglycan (GAG) Cuprolinic blue positively stained the glycosaminoglycan (GAG) side chains of proteoglycans (PGs) with a dark contrast for transmission electron microscopy imaging. At E12, PGs (black arrows) initially associated with the surface of the mesenchymal cells (white asterisks). With increased development (E13) the PGs (black arrows) were found around cells (white asterisks) and independently within the extracellular matrix space. Some cell processes show regular attachment of PGs transverse to the direction of the process (white arrows). At E14, PGs (black arrow) were additionally found to accumulate posterior to the corneal epithelium (white asterisk), affiliated with the basal lamina. At E16, PGs were seen to lie between the cell (white asterisk) and collagen fibrils, and between adjacent collagen fibrils (black arrows).  Epithelium KS:DS = 1.53 Water = 3.04/g dry wt. KS:DS = 2.23 Water = 3.85/g dry wt. Endothelium Figure 4.15 (A) The regional differences in the corneal stroma for the proportion of the two types of corneal GAGs and the water-absorbing properties in these regions are shown. (B) Diagram of the metabolic pathways for dermatan sulfate and keratan sulfate production. The supply of oxygen is the primary factor determining whether dermatan sulfate is made through an aerobic pathway or whether keratan sulfate is made through