Hemidesmosomes Research Articles

Comparative study of the dynamics of focal contacts in live epithelial and mesenchymal cells

To further characterize the morphology and dynamics of focal contacts (FCs) in epithelial cells, we compared the size, number, localization, velocity, and turnover of FCs in epithelial and mesenchymal cell lines. Using immunocytochemistry, we found there were no significant differences between mesenchymal and epithelial cells in number and appearance whereas the location and size of FCs in each cell were different between mesenchymal and epithelial cells. FCs in mesenchymal cells localized at the cell periphery and cell center, but FCs were found only at the cell periphery in epithelial cells. The size of FCs in epithelial cells were significantly smaller than in mesenchymal cells. Next, we compared the dynamics of FCs in both mesenchymal and epithelial cells and found no significant difference between the two groups. Finally, we added inhibitors for the hemidesmosome (HD) proteins, α6 integrin and β4 integrin, to HaCat cell (epithelial) cultures and examined the number and size of FCs. Under these conditions, the size and localization of FCs in HaCat cells became comparable to that of mesenchymal cells. Therefore, we concluded the size and localization of FCs is regulated by the existence of HDs in epithelial cells.

The human corneal epithelium after alcohol delamination: a structural and ultrastructural study

Dilute alcohol is one of the most popular methods for corneal epithelial removal during photorefractive keratectomy (PRK) and laser subepithelial keratomilieusis (LASEK). Even if the technique is used by nearly fifteen years, no concordant data are available on the effects of the exposition to dilute alcohol on the corneal epithelium. As in LASEK the epithelial flap obtained by the previous delamination is repositioned to improve corneal recovery, aim of the present work was to investigate the structure and the ultrastructure of the corneal epithelium after alcohol delamination. Ten patients undergoing PRK for myopic correction were consecutively included in the study. A 9-mm diameter cone was placed on the anaesthetized cornea and it was filled with 25% ethanol in BSS for 25 seconds. The cone was emptied and the corneal surface was washed off with BSS. The epithelial layer was lifted with beaver blade, peeled off with forceps, and processed for light (LM) and transmission electron microscopy (TEM). With LM, whilst superficial and wing cells showed a normal appearance, basal cells had significantly different staining patterns. In fact, in the same microscopic field they showed either normal morphology or paler nuclei and cytoplasm. When the specimens were observed with the TEM, all epithelial cells showed well-preserved intercellular spaces and junctional complexes. In the superficial cells perinuclear vacuolizations were present, whilst wing cells demonstrated no evident morphological changes. Clear basal cells had roundish nuclei with pale chromatin and clear cytoplasm with perinuclear endoplasmic reticulum and mitochondria and a large number of tonofilaments. Their basal membrane was generally intact, with many hemidesmosomes adhering to the basement membrane, which was formed only by the laminae lucida and densa. Dark basal cells showed irregular nuclei with condensed chromatin, vacuolated cytoplasm and few basal hemidesmosomes. Alcohol debridement can be considered as a valuable technique for removing corneal epithelium before PRK or for preparing an epithelial flap before LASEK: in fact it affects the binding of hemidesmosomes to the underlying basement membrane so that the lamina densa is separated from the lamina fibroreticularis. However, the observation of structural and ultrastructural changes of the basal cells, similar to those demonstrated in epithelial flaps obtained with the epikeratome, indicates the need for further studies to evaluate the corneal toxicity of the ethanol.

EGF-induced MAPK Signaling Inhibits Hemidesmosome Formation through Phosphorylation of the Integrin β4

Migration of keratinocytes requires a regulated and dynamic turnover of hemidesmosomes (HDs). We and others have previously identified three serine residues on the integrin β4 cytoplasmic domain that play a critical role in the regulation of HD disassembly. In this study we show that only two of these residues (Ser-1356 and Ser-1364) are phosphorylated in keratinocytes after stimulation with either PMA or EGF. Furthermore, in direct contrast to previous studies performed in vitro, we found that the PMA- and EGF-stimulated phosphorylation of β4 is not mediated by PKC, but by ERK1/2 and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation of β4 increased keratinocyte migration, and reduced the number of stable HDs. Furthermore, mutation of the two serines in β4 to phospho-mimicking aspartic acid decreased its interaction with the cytoskeletal linker protein plectin, as well as the strength of α6β4-mediated adhesion to laminin-332. During mitotic cell rounding, when the overall cell-substrate area is decreased and the number of HDs is reduced, β4 was only phosphorylated on Ser-1356 by a distinct, yet unidentified, kinase. Collectively, these data demonstrate an important role of β4 phosphorylation on residues Ser-1356 and Ser-1364 in the formation and/or stability of HDs.

Conditional ablation of integrin alpha-6 in mouse epidermis leads to skin fragility and inflammation

Hemidesmosomes (HDs) are essential anchorage junctions which mediate the firm attachment of epithelia to the underlying basement membranes, of which one main component is the integrin α6β4. These specific junctions are also able to trigger signalling pathways, via the recruitment and interactions of signalling molecules with HD components such as the cytoplasmic tail of the β4 integrin or the plakin plectin. HDs must also assemble and disassemble depending on the tissue context for example during tissue remodelling. Alterations of HD components or their loss result in skin blistering disorders known as epidermolysis bullosa. Since mice lacking integrin α6 die at birth with severe skin blistering, we have produced a mouse line in which epidermal deletion of integrin α6 can be controlled by tamoxifen injection. We observed that the deletion was mosaic, but that hairless skin such as ears, tails and paws were affected and showed chronic inflammation associated with hyperproliferation, and expression of laminin-111. Interestingly, two cytokines, amphiregulin and epiregulin, previously found increased in integrin α6 deficient cultured keratinocytes, were also increased here in the affected skin. In detached areas, we validate clearly that the absence of integrin α6 leads to a delocalisation of plectin, and the complete disappearance of HD structures.

Ultrastructural and immunohistochemical studies of regenerating epithelium-stroma junctions of rat tracheal epithelium after mechanical injury

Regeneration of the rat tracheal epithelium following mechanical injury was studied by electron microscopy and immunohistochemistry. The first event that occurred following the injury was rapid flattening of the viable epithelial cells of the wound margin and their migration to the injured area. In the early stage, the regenerating epithelium changed from a simple squamous to a multilayered structure, or typical squamous metaplasia. Immunohistochemically, keratin was detected in the regenerating cells, and actin was present linearly along the basal cell membrane of the migrating epithelial cells. Fibronectin also appeared in the wounded stroma immediately beneath the migrating epithelium until the migration was completed. Both laminin and type IV collagen reappeared under the regenerating epithelium from the wound margin after the migration was completed in the middle stage. Ultrastructurally, fibronexus-like structures and so-called anchoring filaments appeared in the epithelium-stroma junction before hemidesmosomes, basal lamina and anchoring fibrils reappeared. We consider that both fibronexus-like structures and anchoring filaments are responsible for the initial adhesion of the regenerating epithelium to the underlying connective tissue.

Collagen XVII

Collagen XVII has been identified as having a role in inherited junctional epidermolysis bullosa non-Herlitz (JEB-other, MIM #226650). The role of collagen XVII in both autoimmune and genetic blistering disorders demonstrates its relevance to dermal-epidermal adhesion. Collagen XVII is a major structural component of the hemidesmosome (HD), a highly specialized multiprotein complex that mediates the anchorage of basal epithelial cells to the underlying basement membrane in stratified, pseudostratified, and transitional epithelia. This article examines the genetic and pathological features of collagen XVII.

Viability of corneal epithelial cells and cleavage of corneal basement after ethanol effect

To evaluate the viability of corneal epithelial cells and to determine the anatomic cleavage on the epithelial basement membrane after various exposure times to 20% ethanol during epithelial flap preparation in laser-assisted subepithelial keratectomy (LASEK) in cadaver eyes. Six human cadaver eyes were exposed to 20% ethanol for 20, 30 and 40 seconds (2 eyes for each group), and another one eye was used as the control. PCNA staining was performed to determine the viability of corneal epithelial cells. Immunofluorescence staining using monoclonal antibodies against collagen VII, and immunohistological staining using monoclonal antibodies against laminin were performed to detect the anatomic location of the cleavage plane on the corneal epithelial flaps created by 20 seconds exposure to 20% ethanol in cadaver eyes. Hematoxylin and eosin staining of epithelial flaps revealed a coherent stratified epithelium. The PCNA positive rates of the epithelial cells in the flap decreased in the 20-second group, 30-second group and 40-second group successively. Immunohistological staining to laminin was patchy in the lifted flap and the remaining corneal basement membrane. Immunofluorescence to collagen VII, the main component of anchoring fibrils remained exclusively in the corneal bed. Viability of the epithelial flap decreased with longer time exposure to ethanol. The cleavage plane of the ethanol-treated corneal epithelial flap is located between the lamina lucida and the lamina densa of the basement membrane where laminin forms hemidesmosome.

Phosphorylation of a novel site on the {beta}4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelial cells to the basement membrane. HD components include the alpha6beta4 integrin, plectin, and BPAGs (bullous pemphigoid antigens). HD disassembly in keratinocytes is necessary for cells to migrate and can be induced by EGF through beta4 integrin phosphorylation. We have identified a novel phosphorylation site on the beta4 integrin: S(1424). Preventing phosphorylation by mutating S-->A(1424) results in increased incorporation of beta4 into HDs and resistance to EGF-induced disassembly. In contrast, mutating S-->D(1424) (mimicking phosphorylation) partially mobilizes beta4 from HDs and potentiates the disassembly effects of other phosphorylation sites. In contrast to previously described sites that are phosphorylated upon growth factor stimulation, S(1424) already exhibits high constitutive phosphorylation, suggesting additional functions. Constitutive phosphorylation of S(1424) is distinctively enriched at the trailing edge of migrating keratinocytes where HDs are disassembled. Although most of this S(1424)-phosphorylated beta4 is found dissociated from HDs, a substantial amount can be associated with HDs near the cell margins, colocalizing with plectin but always excluding BPAGs, suggesting that phospho-S(1424) might be a mechanism to dissociate beta4 from BPAGs. S(1424) phosphorylation is PKC dependent. These data suggest an important role for S(1424) in the gradual disassembly of HDs induced by cell retraction.

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea

Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease. We employed thin-section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages. The layering of the zebrafish cornea into an epithelium, a Bowman's layer, stroma and endothelium was observed starting at 72 hr post-fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2-week-old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened. The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.

Serine Phosphorylation of the Integrin β4 Subunit Is Necessary for Epidermal Growth Factor Receptor–induced Hemidesmosome Disruption

Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of alpha6beta4 to plectin plays a central role in their assembly. We have defined three regions on beta4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of beta4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of beta4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the beta4 subunit.

Multiple Functions of the Integrin α6β4 in Epidermal Homeostasis and Tumorigenesis

Since the discovery of the α6β4 integrin in the late 1980s, our understanding of its role in providing stable adhesion of epithelial cells to basement membranes (BM) has significantly increased. α6β4 plays a key role in the formation and stabilization of junctional adhesion complexes called hemidesmosomes (HDs) that are connected to the intermediate filament (IF) system, as well as in the regulation of a variety of signaling processes. However, it is not clear as yet whether α6β4 participates in cell signaling by serving as a substrate for tyrosine kinases and as an adaptor for their associated signaling proteins or whether its role in cellular processes is passive, involving regulation of the assembly and disassembly of HDs. In this review, we will discuss the roles attributed to α6β4 and the controversies in the field.

Colocalization of Multiple Laminin Isoforms Predominantly beneath Hemidesmosomes in the Upper Lamina Densa of the Epidermal Basement Membrane

Multiple laminin isoforms including laminins 5 (alpha3 beta3 gamma2), 6 (alpha3 beta1 gamma1), 10 (alpha5 beta1 gamma1), and possibly laminins 7 (alpha3 beta2 gamma1) and 11 (alpha5 beta2 gamma1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 (alpha3 beta3 gamma2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the alpha5, beta1, beta2, and gamma1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n=15) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the alpha5, beta1, and gamma1 chains (present in laminins 10/11) and beta2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n=6). In the basement membrane of human epidermis, the alpha5, beta1, beta2, and gamma1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the alpha5, beta1, beta2, and gamma1 chains to the upper lamina densa, with between 84% and 92% of labeling restricted to beneath the HDs, similar to laminin 5 (n> or =200 gold particles per sample, sample number n=4) but distinct from collagen IV labeling (with only 63% labeling beneath HDs, p<0.001). Taken together, the majority of the alpha5beta1/beta2gamma1 laminin chains are located beneath HDs. This suggests that laminin-10-associated chains have specific functions or molecular interactions beneath HDs in the epidermal basement membrane.

Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm alpha3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the alpha3 G domain processing in physiological conditions, we have expressed a series of mutant alpha3 complementary DNA in human primary alpha3-null keratinocytes immortalized by human papillomavirus E6E7 (HKalpha3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the alpha3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of alpha3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKalpha3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5.

Disruption of ERBB2IP Is not Associated with Dystrophic Epidermolysis Bullosa in Both Father and Son Carrying a Balanced 5;13 Translocation

Mutations in the type VII collagen gene (COL7A1) cause autosomal recessive and autosomal dominant inherited dystrophic epidermolysis bullosa (DEB). We report a family with three individuals who present blistering, scarring, hypo- and hyperpigmentation, and nail dystrophy suggestive for DEB. Whereas father and son carry a 5;13 translocation, the daughter shows a normal karyotype. Segregation analysis revealed that all affected family members inherited the same COL7A1 allele. Mutation analysis disclosed a heterozygous missense mutation, c.6227G > A (p.G2076D), in COL7A1 in all affected individuals. Delineation of the translocation breakpoints showed that the ERBB2IP (erbb2 interacting protein or Erbin) gene is disrupted in 5q13.1 and GPC6 in 13q32. GPC6 encodes glypican 6 belonging to a family of cell surface heparan sulfate proteoglycans. The binding partners of Erbin, BP230 (BPAG1) and the integrin beta4 subunit, both involved in hemidesmosome (HD) function, and the presence of Erbin in HD suggested that it plays a role in establishment and maintenance of cell-basement membrane adhesions. However, loss of function of one ERBB2IP copy or expression of a putative novel ERBB2IP fusion protein did not apparently modulate the DEB phenotype in both translocation patients. Nonetheless, one cannot yet exclude that ERBB2IP is a candidate for human blistering disorders such as epidermolysis bullosa.

Both type-I hemidesmosomes and adherens-type junctions contribute to the cell–substratum adhesion system in myoepithelial cells

Myoepithelial cells present in exocrine glands cause secretion from the glands by contraction. They have mixed characteristics with regard to cytoskeletal elements, containing both epithelial-type intermediate filaments and smooth muscle-type myofilaments. For further characterization, myoepithelial cells from bovine apocrine sweat glands and tracheal glands were here examined with special attention to the cell–substratum adhesion system. Immunofluorescence microscopy using a panel of antibodies against adherens-type junctional and hemidesmosomal proteins demonstrated two types of cell–substratum junctions in myoepithelial cells from both glands. Type-I hemidesmosomes (HDs) consisting of plectin, BP230, integrin α6 β4, and BP180 were thus observed as punctate arrays longitudinally arranged along myoepithelial cell surfaces, while adherens-type junctions were similarly evident as linear rib-like structures. Double-label immunofluoresence revealed the two junctions to be distributed in a mutually exclusive or independent manner. Electron microscopy further demonstrated that apocrine myoepithelial cells surround secretory epithelial cells completely, without any gaps, HDs being abundant along the basement membrane, but with no distinct structures in the inter-hemidesmosomal regions. Immunoelectron microscopy, however, revealed an inter-hemidesmosomal localization of vinculin, pointing to the existence of adherens-type junctions. Secretory epithelial cells in tracheal glands were found not to be completely covered with myoepithelial cells, so that more than half of them are directly attached to the basement membrane, where they form type II-HDs lacking BP230 and BP180, but no detectable adherens junctions, like epidermal basal cells and sebaceous gland cells. These observations demonstrate that, in addition to their cytoskeleton, myoepithelial cells have both epithelial- and smooth muscle-type cell–substratum adhesion structures, i.e. HDs and dense plaque-like adherens junctions.

Remodeling of desmosomal and hemidesmosomal adhesion systems during human hair follicle development

Complex cell–cell and cell–extracellular matrix interactions are implied in the induction and promotion of hair follicle morphogenesis. Desmosomes (DS) and hemidesmosomes (HD) are thought to play important roles in the signal transduction during the regulation of cell growth and differentiation. Recently, remodeling of these two cell adhesion systems was suggested to underlie hair follicle morphogenesis in mouse pelage hair follicles [1]. Mindful of this hypothesis, we speculated that remodeling of DS and HD are involved in the regulation of cell growth and differentiation during human hair follicle morphogenesis. In the present study, we investigated the expression of major DS and HD components and the ultrastructural formation of DS and HD in the developing human hair follicles of skin samples from human fetuses from a series of estimated gestational ages (EGA) (49–163 days EGA).

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study.

Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro.

Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM). Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension. Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload). Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.

Anatomy and physiology of the inner hoof wall

The equine hoof capsule protects the softer, more sensitive, structures within. Failure of the connection between hoof and bone (the lamellar, distal phalangeal attachment apparatus) results in the crippling lameness of laminitis. Dozens of different keratin molecules, with differing biomechanical properties and molecular weights, with varying degrees of hardness and sulfur concentration, are expressed in hoof tissue. Nonstop production of new hoof makes good the continual loss of hoof wall, occurring at the distal ground surface. A new calculation of the proliferative index (PI), for basal cells of the coronet and lamellae of the dorsal hoof wall, shows that active basal cell proliferation occurs only in the coronet and proximal lamellae. The remaining lamellae are virtually nonproliferative and remodeling within the hoof wall lamellae, that must occur as the hoof wall moves past the stationary distal phalanx, is a function of the controlled activation and inhibition of matrix metalloproteinases (MMPs). Proliferating coronet basal cells organize into thin, elongated cylinders (hoof wall tubules) approximately 0.2 mm in diameter. The central hollow medulla of each tubule comprises about 2% of the total hoof wall volume at the toe. The tubules of the s tratum medium are arranged into zones based on the density of tubules in the intertubular horn. This tubule density gradient appears to be a mechanism for smooth energy transfer, from the rigid (high tubule density) outer wall to the more plastic (low tubule density) inner wall, and ultimately to the distal phalanx. The gradient in tubule density mirrors the gradient in water content across the hoof wall and together these factors represent an optimum design for equine hoof wall. The lamellar corium derives most of its blood supply from the branches of the terminal arch which perforates the distal phalanx. Numerous anastomoses form an arterial lattice beneath and between the epidermal lamellae and blood can flow proximally to the coronary circumflex artery and distally to the solar circumflex artery. Veins within the foot are valveless and this can be exploited clinically for retrograde venous therapy or contrast radiography (venograms). In the lamellar dermal microcirculation numerous (500/cm 2 ) arteriovenous anastomoses connect the axial arteries to axial veins; their normal role relates to thermoregulation and pressure modulation. On the inner hoof wall ( stratum internum ) is the stratum lamellatum (layer of leaves) is named after the 550 to 600 epidermal lamellae (primary epidermal lamellae or PEL) which project from the surface of the inner hoof wall ( stratum internum ) in parallel rows. Each PEL has 150 to 200 secondary lamellae on its surface and normal SELs have a constant histological appearance that only laminitis alters. The basement membrane (BM), a tough, unbroken sheet of extracellular matrix, forms the interface between the lamellar epidermis and the dermis. It is a three-dimensional sheet of fine, anastomosing cords consisting of filaments of collagen IV ensheathed with glycoproteins, in particular laminins 1 and 5. The plasma membrane of each lamellar basal cell is attached to the BM by numerous electron dense adhesion plaques or hemidesmosomes (HDs). The various proteins of each HD occur on both sides of the basal cell plasmalemma and with anchoring filaments (AFs) form a bridge linking the interior of the basal cell to the exterior connective tissue and ultimately the distal phalanx. Importantly, HDs are maintained and assembled by glucose-consuming phosphorylation reactions and perturbations of glucose metabolism may be involved in laminitis pathogenesis. HDs and AFs are essential to normal hoof lamellar integrity and this is illustrated by horses that inherit mutations in the genes expressing these proteins. Failure to correctly express plectin or laminin5 (HD and AF proteins, respectively) results in neonatal, laminitis-like pathology. Laminitis destroys the BM and its components and without an intact, functional, basement membrane, the structure and function of the lamellar epidermis is pathologically compromised. Transcription and activation of MMP-2 occurs in normal hoof lamellae and in increased amounts during laminitis. Accidental triggering of uncontrolled, excessive MMP production of MMPs, present in the cells of the secondary epidermal lamellae, presumably for normal remodeling purposes, appears to be an important player in laminitis pathogenesis.

JID Abstracts

Laminins comprise a family of over 20 extracellular glycoproteins that are involved in cell-basement membrane zone (BMZ) adhesion. In skin, laminin isoforms are present in the epidermal BMZ, around blood vessels (BVs), nerves and adnexal structures. Recently, specific antibodies have become available that recognize laminin chains. The antibody 4C7 recognizes the terminal globular domain of the laminin α5 chain in laminins 10 (α5, β1, γ1) and 11 (α5, β2, γ1). Laminin 5 (α3, β3, γ2) is a well-studied hemidesmosome (HD)-associated laminin. We therefore compared the α5 chain expression with laminin 5 in normal and diseased skin. Laminin α3 and α5 chains were linearly expressed in the epidermal BMZ, except beneath melanocytes. The α5 chain but not the α3 chain was expressed around BVs in adult skin. Late anagen hair follicles stained with anti-α3 and α5 chain antibodies showed typical expression patterns described for HD antigens. In cultured RAC-11P bladder cells, that assemble HD-like structures in vitro, both α3 and α5 chains colocalized to discrete focal areas beneath sub-confluent cells presumed to be HDs. Finally, immunogold electron microscopy localized the α5 terminal globular domain to the lower lamina lucida (n=200 gold particles). The majority (75%) of α5 chain labeling was restricted to beneath HDs, similar to laminin 5 (77% labeling restricted to HDs). Laminin α3 and α5 chain expression was assessed in control (n=4) and patients skin suffering from epidermolysis bullosa (EB, n=11). Laminin α5 chain expression was normal in control and all EB subtypes. This was in contrast to the α3 chain that was reduced or absent in JEB subtypes. These data suggest that the α5 chain exhibits an expression pattern restricted to beneath HDs, similar to the α3 chain of laminin 5, however the stable expression of α5 chains is independent of laminin 5. This further supports the hypothesis that several laminin variants may covalently assemble as a multi-molecular complex that together promotes stable epithelial-BMZ attachment.