OP: Corneal Epithelium Physiology

What is the extracellular matrix? What are the 7 cell/tissue processes that it performs? Lastly, what are the two categories and what are two examples of each?

• Dynamic 3D network of macromolecules that provide structural support for cells and tissues

• Regulates the following functions:

  • Proliferation

  • Migration

  • Differentiation

  • Angiogenesis

  • Immune function

  • Autophagy

  • Tissue separation/shaping

• Pericellular matrix

  • Basement membrane of epithelia

  • Glycocalyx

• Interstitial matrix

  • Cartliage/bone

  • Stroma of cornea

What are the 8 major components of the ECM?

1. Proteoglycans

2. Collagens

3. Matrix metalloproteinases (MMPs)

4. Elastic fibers (fibrillin, elastin)

5. Lysyl oxidase

6. Laminin

7. Tenascin

8. Fibronectin

What are the 3 components of the cornea epithelial basement membrane? What is each for?

• Laminin

  • Principle component of lamina lucida

• Collage IV

  • Principle component of lamina densa

• Collage type VII

  • Anchoring fibrils

Where are hemidesmosomes found, what is their function, and what three things are they comprised of? What is the purpose of each of these components?

• Cellular structure found in basal membrane of corneal epithelial cells

• Adheres BM to cells

1. Intermediate filaments

   • Structural cytoskeletal component

   • Strengthens hemidesmosomes

2. Plectin

   • Forms intracellular plaque

   • Binds to intermediate filament of cytoskeleton

   • Binds to integrins

3. Integrins

   • Transmembrane proteins

   • Binds to laminin

   • Binds to plectin

How many subunits does collagen IV form? What human made structure does this mimic? Lastly, what does it do and why?

• Forms a molecular subunit of four proteins

• Chicken wire like structure

• Interconnects many proteins

• Provides strength

What are corneal erosions, what is a common presentation with it, and what are the 4 different causes? Explain what the genetic causes affect and present with.

• Failure of corneal epithelial cells to adhere to BM/Bowman’s

• Induces pain due to exposed nerve endings

• Corneal injury

• Disruption of molecular connection between hemidesmosomes and the basement membrane (EBMD)

• Genetic predispositions:

  • Alport’s syndrome

    • Mutations in collagen IV

    • Epithelial cell death + cataracts

  • Epidermolysis bullosa

    • Disruptions in other hemidesmosomal/basement membrane genes

What is epithelial basement membrane dystrophy also known as? What is the clinical finding and the mechanism behind it?

• Cogan’s microcytic epithelial dystrophy or map-dot-fingerprint dystrophy

• Geographic map-like lines and subepithelial microcysts

• Abnormal thickening of basement membrane and intrustion of the basement membrane into more superficial layers of cornea.

What is a risk factor for EBMD? How does it prevalence change with age? What are the symptoms of it and what is it a risk factor for itself? Why is it a risk factor?

• Family history

• 2% of general population, increased risk with age

• Often asymptomatic yet also often an underlying factor for corneal erosions (nearly a third)

  • Cells anterior to the ectopic ECM do not adhere well

  • Consequently biochemically weaker

  • Furthermore, anchoring filaments are not bound to Bowman’s or the stroma

What are the two distinguishing characteristics about stem cells? Furthermore, what are the 2 groups and how do the 3 types fit into those groups? Lastly, state what pluri/multi/unipotent cells can differentiate into (generally).

1. Undifferentiated cells capable of renewing themselves through cell division

2. They have the potential to differentiate into a specific cell type

• Embryonic stem cells (pluri or totipotent)

• Somatic adult stem cells (multipotent)

• Pluripotent: give rise to all tissue cell types

• Multipotent: give rise to multiple cell types

• Unipotent: give rise to a single cell type

What is exfoliation/desquamation in reference to the corneal epithelium? What 4 things can cause it? Furthermore, how do the cells of the cornea move and why? Lastly, roughly how long does it take for complete corneal epithelium turnover?

Cell loss from the epithelium d/t the following factors:

• Constant cell loss via sloughing

• Blinking induced

• Minor abrasions

• Eye rubbing

Centripetal movement of cells

• D/t growth pressure

• Entire corneal epithelium renewed in 9-12 months

Explain X+Y=Z for corneal epithelium.

X = cell contribution d/t basal cell proliferation (mitosis)

Y = cell contribution due to centripetal movement of cells from peripheral (limbus) to central cornea

Z = normal loss of cells from the corneal surface

What are the two conflicting hypotheses of corneal epithelium turnover? Explain where the stem cells are found in each hypothesis, how transient amplifying cells move, and what refreshes the epithelium during homeostasis/injury?

LESC

• Stem cells found exclusively in limbus

• TACs move/divide centripetally

• Refresh corneal epithelium during homeostasis and injury

Corneal epithelial stem cell

• Stem cells are found in limbus and basal cornea epithelium

• TAC’s move/divide centrifugally

• LESC’s contribute only during injury

• CESCs contribute during homeostasis and injury

Where is the source of corneal epithelial cells?

Limbal epithelial thickenings known as palisades of Vogt

What are the 5 characteristics of limbal stem cells?

1. Low mitotic activity

2. Multipotent

3. Non-differentiated when in limbal epithelium

   • Cornea specific keratins absent

4. Long life span

5. Unlimited potential for cell division

What 2 cells does cell division of LSCs produce? How is the mitotic rate affected of one of these cells?

1. One remains a LSC

2. One becomes a TAC (transient amplifying cell)

Mitosis for the TAC rapidly increases, especially in the periphery.

What 2 ways do TACs migrate? Explain how each is migration is produced.

1. Centripetal

   • Undergo multiple rounds of replication during centripetal movement

   • Progressively lose multipotency with each division

2. Anterior

   • Post-mitotic and thus stops dividing

What is a stem cell niche, where is the limbus stem cell niche, and what are the 6 factors that help it foster multipotency of those stem cells?

• A region that protects a stem cell population and helps maintain their multipotency

• Epithelial thickenings in the limbus function as this niche (palisades of Vogt)

• Multipotency factors:

  • Thickness of epithelium

  • Rich in melanin

  • Different array of basement membrane proteins than central cornea

  • Distinct ECM can promote stemness

  • Distinct ECM can promote differentiation

  • Nearby blood vessels for O2, growth factors and antioxidants

Where are corneal stroma stem cells located, what nutures them, how does it fit the definition of a stem cell, and what particularly valuable cell can they differentiate into in the corneal stroma?

• Subjacent to limbal basal cells

• Limbal stem cell niche nutures it

• Fits the definition of stem cell because it can self renew and is multipotent

• Can differentiate to form keratocytes

Where are conj stem cells located, where are they concentrated, what two cells do they often differentiate into, and explain how they are distinct from limbal stem cells.

• Unknown location

• Concentrated in fornical conjunctiva and spread throughout

• Multipotency allows them to differentiate into epithelial and goblet cells

• Distinct from LSCs as they do not produce proteins that are corneal specific

What is classified as limbal stem cell deficiency? What are the 7 presentations? What are the 6 etiologies?

• Decreased ability to repopulate corneal epithelium caused by any process that diminishes the supply of stem cells or disrupts their niche

• Presentations:

  • Blurry vision

  • Foreign-body sensation

  • Photophobia

  • Tearing

  • Pain

  • Loss of palisades of Vogt

  • Cornea epithelial thinning

• Caused by autoimmune disorders, chemical/thermal injury, contact lenses, surgical damage, infections, and in congenital malformations.

What tissues of the eye does ocular surface squamous neoplasia affect? What region is particularly susceptible? What are the 3 risk factors? What is its range of severity? Lastly, what cells does it affect?

• Affects cornea and/or conj

• Conj of limbus region is particularly susceptible

• Risk factors

  • UV

  • HIV

  • HPV

• Ranges from mild dysplasia to invasive squamos cell carcinoma

• Affects basal limbal stem cells

What tissue overgrows its bounds in pterygiums and where does it overgrow? What layer degrades in a pterygium? What other repair response does it resemble? Furthermore, what are the two possible etiologies? Lastly, what acts as a physical barrier to the tissue that overgrows its bounds in a pterygium?

• encroachment of bulbar conj onto the cornea

• degradation of bowman’s layer

• resembles aberrant wound healing response

• two potential etiologies

  • mutation of limbal stem/epithelial cells causing them to inappropriately proliferate/differentiate into conj cells

  • destruction of limbal stem/epithelial cells which normally act as a physical barrier to conj proliferation and encroachment into the cornea (a local limbal stem cell deficiency)

What are the differences between OSSN and pterygium? Touch on what tissues each involve, the vessel pattern each exhibit, which processes each trigger, and the pathogenic potential of each.

OSSN:

• Variably involves growth of conj, limbal/cornea epithelia

• zigzag vessel pattern

• surface keratinization

• potential to become invasive/malignant

Pterygium:

• Growth of subepithelial conj fibroblastic tissue over the cornea

  • Underlying stroma of activated fibroblasts

  • Neovascularization (straight vessel patterns)

  • ECM remodeling

  • Inflammation

  • Leading edge of altered limbal epithelial cells

  • Goblet cell hyperplasia

What is the cause of a pinguecula? What does it cause inappropriate production of? What can this lead to? Where does it occur? What is its appearance? What do advanced stages increase the risk of disrupting? What can this disruption cause within the affected cells? Lastly, what can this lead to and what can pingueculas occur in tandem with?

• UV damage of fibroblasts beneath epithelium

  • Cause inappropriate production of ECM proteins (elastin)

  • Can lead to increase in fibroblast cell number

• Occurs in bulbar conj and limbus

• Appears as a creamy colored, chalky growth on conj surface

• Advanced stages make UV damage of cells more likely by disrupting protective mechanisms

  • Can cause mutation of DNA in limbal stem/epithelial cells

  • Leads to inappropriate growth

  • Pterygium and pinguecula can occur together

What 3 things does the amount and rate of healing with corneal injuries depend on? Roughly how long does it take for superficial abrasions to heal? What about deeper injuries within the stroma?

• Amount/rate of healing and scarring depends on depth of abrasion, cause, and tear quality

• Small superficial abrasions heal within 1-2 days

• Deeper injuries take up to 2 years to fully heal

What are the 4 stages of corneal epithelium wound repair? How long after a wound does stage 1 occur? What is the duration of stage 2 and 3 dependent on?

Stage 1: Latent phase (4-6 hours after wound)

Stage 2: Cell migration (duration dependent on wound size)

Stage 3: Epithelial mitosis (duration dependent on wound size)

Stage 4: Reassembly of adhesive contacts

What is stage 1 of epithelial wound repair known as? What 3 things happen to the epithelial cells proximal to the wound? What are the 2 substances released as signals of cellular stress during this stage? What about the 2 substances that cause nerve damage? What induces the degradation of the basement membrane components and what specific components does it affect? Furthermore, what 3 things removed damaged cells and what is inhibited at the end of this phase?

Latent phase:

• Epithelial cells near wound change morphology

  • Loss of surface microplicae

  • Rounding and retraction of epithelial cells at wound edge

  • Basal cells flatten

• Signaling/Chemotaxis

  • Ca2+ (cellular stress)

  • H2O2 (cellular stress)

  • Cytokines (nerve damage)

  • Substance P (nerve damage)

• Degradation of basement membrane components

  • Hemidesmosomal attachments between basement membrane and basal cells disappear outward from wound

  • This process is facilitated by PMN/neutrophils

• Damaged cell removal

  • Tear flush

  • Leukocyte phagocytic activity

  • Apoptosis of damaged cells

• Mitosis inhibited

How do PMNs get into the tears? How about the stroma? Lastly, what do they facilitate?

• Infiltrate tears from lymphoid follicles and diffuse lymphoid tissue beneath conjunctiva

• Infiltrate the stroma from limbal blood vessels upon corneal abrasion to the site of the wound

• Facilitate events of latent phase

What is stage 2 of epithelial wound repair known as? What are the 2 cellular processes and where do they each extend to? What is this driven by and what two micromolecules allow for this? Lastly, what does this assist in regards to wound repair and what does it utilize in order to extend?

Cellular processes:

• Lamellipodia (broad)

• Filopodia (narrow)

• Extend from margin of epithelial wound

• Driven by cytoskeleton polymerization

  • Actin

  • Myosin

• Assist cell migration across the wound area covering the region of cell loss

• Use focal adhesions

How do filopodia form?

Pushing out from plasmalemma utilizing actin

What does actin bind with in cellular migration and what does this also bind?

Actin binds focal adhesions which bind ECM

What are focal adhesions, what are they utilized for, and what are the 2 components of them?

• Protein complex utilized to connect the intracellular cytoskeleton with the ECM

  • Transmembrane integrins

  • Actomyosin

What is stage 3 of epithelial wound repair known as? What occurs during this stage?

Epithelial cell proliferation

• Mitosis and differentiation of transit amplifying cells (TACs) resumes until full thickness is restored

What is stage 4 of epithelial wound repair known as? What is re-established, what 2 things are synthesized, how long can it take, and what are you prone to and for how long following an epithelial wound?

Reassembly of adhesive structures

• Superficial corneal cell tight-junctions is re-established

• Synthesis of hemidesmosomes and anchoring filaments

• 36 hours to several months

• Until anchoring filaments are restablished you are still prone to erosions which can take weeks

How does diabetes affect corneal wound healing? What changes in regard to water can it cause in the cornea? What layer can it alter? Furthermore, what is glucose’s affect on EGF signaling, adhesions to ECM, and nerves? What can glucose be converted into and what does an excessive amount of this product cause? Lastly, which pump does this affect, how does it affect the pumps, and what abnormalities can it cause?

• decreased wound healing

• corneal edema

• altered epithelial basement membrane

• increased glucose causes:

  • Inhibition of EGF signaling

  • Abnormal adhesions to ECM during epithelial migration

  • Long term affects on nerves (nerves promote healing too)

  • Conversion into sorbitol

  • Excess sorbitol prevents healing of corneal epithelium

  • NKA inhibition

  • Decreased pump function

  • Morphological abnormalities of endothelial cells

  • Sorbitols high negative charge may also contribute to influx of water

What is the first step of corneal stroma healing? What happens to the keratocytes within the wound? What is this facilitated by and how are they handled?

• Epithelial healing proceeds to cover and fill in the stromal abrasion

• They undergo apoptosis

• Facilitated by PMNs

• Cleared by macrophages

After epithelial migration during corneal stroma repair, what do nearby keratocytes do? What region do they form and between where? Furthermore, what two things does this produce? Lastly, what is notable about the injured region at this stage and what infiltrates during it?

• Nearby viable keratocytes differentiate into hypercellular myofibroblasts

  • Proliferate and form a region of hypercellularity between recently migrated epithelium and remaining stroma

  • Produce sparse ECM with some GAGs

  • Produce alpha smooth muscle actin (migratory protein)

  • Injured region is not transparent

• Infiltration of immune cells

What two cell types do the hypercellular myofibroblasts differentiate into? What 2 things does the first type produce? What is the organization of the resulting fibrils and the appearance of the stroma? Furthermore, what 2 things does the second type produce? What is notably absent from their production and what does this help facilitate? Lastly, what cellular process can occur that aids in transparency?

• Myofibroblasts

  • Produce alpha smooth muscle actin

  • Produce high levels of collagen & hyaluronan but low levels of mature stromal GAGs

  • Resulting fibrils are disorganized and stroma is opaque

• Wound fibroblasts

  • Produce high levels of collagen and mature stromal GAGs to form highly organized fibril structure

  • Do not produce alpha smooth muscle actin

  • Facilitate transparency

• Myofibroblasts can convert to wound fibroblasts to restore transparency over time

What happens to Bowman’s in the event of an injury?

• Bowman’s layer does not heal, if damaged it’s absence is permanent

What relative percent of total cells are immune cells in the stroma? Furthermore, what two cell types are present and where do they tend to be located?

• Macrophages

  • tend to be located in basal stroma

• Dendritic cells

  • Anterior stroma and epithelium

• Small % of total cells

What type of cell are dendritic cells, what do they do, and what 2 places do they come from? Furthermore, what 2 places are they found in, where are they more numerous and are they non-moving?

• Antigen presenting cells that phagocytose pathogens and display foreign antigens to T-cells

• Come from myeloid (bone-marrow) and lymphoid lineages (lymph nodes, thymus, tonsil, and spleen)

• Found in corneal epithelium and stroma

• More numerous peripherally than centrally

• Migratory

What cells increase in presence during a corneal inflammatory response?

• There is an increase in density of dendritic cells and macrophages upon inflammatory response due to injury or infection

What causes haze after photorefractive keratectomy?

• Transformations of keratocytes to highly reflective myofibroblasts

What do nerves do in regards to the cellular response in corneal injury? How do impulses travel and what does this induce?

• Nerves contribute to cellular response in corneal injury

• Not only do impulses travel down axon to the parent cell, but also to other non-stimulated branches (in the reverse direction: antidromically)

• Induces the release of neuropeptides (substance P)

What type of NT is substance P? Where is it found? When and where is it released? What does it contribute to? Lastly, what 2 things does it stimulate?

• Substance P

  • Small neuropeptide

  • Found in most TG neurons

  • Released from nerve endings upon noxious stimuli

  • Contributes to local inflammatory response through stimulation of cytokine production

  • Stimulates epithelial mitosis and migration

What is neurotrophic keratopathy? How does it impact corneal sensitivity and epithelial wound healing? What is it caused by and what are the 5 etiologies? What is a drug can treat it? What is the commercial name? What does it function to do and what does it substitute? Lastly, what does it affect in regards to nerves?

• Rare degenerative disease of the cornea

• Reduced corneal sensitivity and impaired epithelial wound healing

• Caused by injury/dysfunction of trigeminal nerve

  • Viral infection

  • Surgery complications

  • Diabetes mellitus

  • Medications

  • Injury

• Active drug: recombinant human NGF (nerve growth factor)/cenegermin

• Commercial name: Oxervate

• Functions to promote signaling pathways that positively effect healing process

• Substitutes for lack of nerves

• Affects nerve growth