Equine digital laminae span between the surface of the distal phalanx and the inner hoof wall. Laminae are composed of 500 to 600 vertical folds of keratinized epidermal tissue (the primary epidermal laminae), which are contiguous with the inner hoof wall, and interdigitated folds of connective tissue (the primary dermal laminae), which are contiguous with the distal phalanx.1 Each fold of primary epidermal and dermal laminae has 150 to 200 interdigitated folds of secondary laminae, which results in a greatly expanded contact area between these tissue layers. The secondary epidermal and dermal laminae join at a basement membrane, which is a meshwork of collagen fibers and laminins cross-linked and anchored via hemidesmosomes to basal epithelial cells residing at the boundary of the epidermal laminae.2 The basement membrane is tethered to tensile collagen fibers of the dermal laminae, which are bound by a variety of ECM components to each other and to integrins expressed by cells on both sides of the membrane,3 which ensures integrity of the 2-layer structure. The basal epithelial cells give rise to keratinocytes that move outward toward the hoof wall and, analogous to skin epidermal cells, increase their keratin content, generate a cornified cell envelope, and undergo terminal differentiation.4,5
The digital laminae resist force imposed by the deep flexor tendon through the distal phalanx to the dermal attachments to the bone.6–8 They also support the vertical load of the horse and accommodate compression and stretch deformation created by the flexing, twisting, and tilting of the hoof capsule under various loading conditions. In addition, the laminae absorb a portion of the concussive shock imposed when the hoof strikes a solid surface. Concussive shock most likely dissipates in the hoof by freedom of movement of hydrated keratin fibers within the epidermal laminae and transfer of load from the hoof wall to the dermal connective tissue and distal phalanx through the basal epithelial cell layer. Therefore, basal epithelial cells are subjected to biomechanical stress and concussion waves of various amplitude and frequency.
Analyses via model systems reveal that cells adapt to constant and discontinuous mechanical stress through mechanoreceptor signaling.9,10 In this regard, chondrocyte explants that are subjected to dynamic compression within a tolerable range respond via elevated production of ECM,9 including elevated production of large polysulfated proteoglycans. Given that equine digital laminae are also subject to dynamic compression or stretching, they may also have a highly specialized ECM that is rich in polysulfated proteoglycans. Indeed, the gene encoding ADAMTS-4, which is a secreted enzyme that regulates turnover of large polysulfated proteoglycans in the ECM, is expressed in laminae of healthy horses,11 which suggests that its substrates may also be present in the tissue.
The main substrates of ADAMTS-4 in peripheral tissues are aggrecan and versican.12 These proteoglycans have GAG attachment domains that can be heavily substituted with chondroitin sulfate side chains and, in the case of aggrecan, keratan sulfate side chains.13–15 Versican is composed of 4 domains: an N-terminal G1 domain, which has the hyaluronan-binding site; an αGAG domain, which has sites for attachment of chondroitin sulfate side chains; a βGAG domain, which has sites for attachment of chondroitin sulfate side chains; and a lectin-like C-terminal domain, which has 2 epidermal growth factor–like motifs.16 There are 4 splice variants (isoforms) of versican: the V0 isoform, which has all 4 domains in the same order; the V1 isoform, which has only G1, βGAG, and C domains; the V2 isoform, which has only G1, αGAG, and C domains; and the V3 isoform, which has only G1 and C domains and lacks GAGs.17
Both aggrecan and versican have a hyaluronan binding site in the N-terminal G1 domain, which facilitates assembly of massive and highly charged macromolecular complexes in the ECM. The anionic groups on the GAGs carry with them positively charged counterions, such as Na+, that create an osmotic gradient and draw water into the tissue. The resulting hydrated gel protects tissue from compression deformation.13,18,19 Given that laminae express the gene encoding ADAMTS-4 and are subjected to biomechanical and concussion stress, it was of interest to determine expression and localization patterns of ADAMTS-4 and its proteoglycan substrates in the laminae. The purpose of the study reported here was to determine whether ADAMTS-4 and its substrates are present in healthy laminae and whether they localize within basal epithelial cells of the secondary epidermal laminae rather than in the ECM.
Materials and Methods
Sample—Laminae of the forelimb hooves were collected from 8 healthy horses as previously described.20,21 Briefly, healthy horses were anesthetized and the distal aspect of the right forelimbs was detached at the metacarpophalangeal joint; horses were euthanized immediately thereafter in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Missouri College of Veterinary Medicine. Excised hoofs were sectioned with a band saw, and segments of lamina (approx 5 × 5 × 5 mm) spanning from the inner hoof wall to the outer surface of the distal phalanx were dissected with a sharp scalpel and flash frozen in liquid nitrogen for molecular and biochemical analyses. For immunofluorescent microscopy, segments were embedded in a commercial preparation of water-soluble glycols and resinsa and frozen over dry ice (6/8 laminae). All segments of laminae were processed or frozen within 5 minutes after detachment of the distal aspect of the right forelimb.
Two female New Zealand White rabbits were vaccinated with an equine ADAMTS-4–specific peptide conjugated to KLH and emulsified in an adjuvant.b The peptide NTPEDSDPDHFD corresponded to amino acids 300 to 311 of the equine ADAMTS-4 sequence and was synthesizedc with an N-terminal cysteine residue to facilitate conjugation to the KLH. The peptide was chosen from an area of ADAMTS-4 predicted to be a loop structure in the metalloproteinase domain on the basis of homology to the human protein sequence, for which the crystal structure has been determined. Briefly, each rabbit received 500 μg of protein dispersed over 10 sites on the dorsum, with an additional 250 μg of peptide-KLH emulsified in an adjuvantd administered 3 times over the course of 10 weeks. The rabbits were exsanguinated 10 days after the last vaccination; serum was harvested and stored at −20°C until use. All procedures were performed under protocols approved by the Institutional Animal Care and Use Committee of the University of Massachusetts.
Peptide-specific antibody was affinity purified from rabbit serum by means of cross-linked polysaccharide polymer beadse conjugated to the NTPEDSDPDHFD peptide. The concentration of purified antibody was determined via spectrophotometry.
RNA extraction—An RNA extraction kitg was used to collect RNA from 3 sections of dorsal lamina from each horse. Briefly, flash-frozen tissue was pulverized in a chilled (on dry ice) biopulverizer,h homogenized in the lysis buffer provided, then passed through the column provided and washed accordingly. The purity and concentration of RNA were determined,f and extracted RNA was used for cDNA preparation only when the 260- to 280-nm absorbance ratio and the 260- to 230-nm absorbance ratio were approximately 2.0. Integrity of isolated RNA was confirmed via electrophoresis on a 1.0% agarose gel and staining with a proprietary polynucleotide gel stain.i
PCR assay—The cDNA was synthesized from isolated RNA with a cDNA synthesis kit.j Primer sets for ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were generated against equine sequences (Appendix 1). Glyceraldehyde 3-phosphate dehydrogenase was used as a housekeeping gene as previously described.11 Briefly, RT-qPCR reactions were performed in accordance with the manufacturer's instruction by use of a proprietary reaction mixture that contained high-performance reverse transcriptase.k Data were determined via a thermal cyclerl as previously described.11 Nonquantitative PCR-specific cDNA fragments of 4 versican isoforms (V0, V1, V2, and V3) were amplified via PCR assay22 with specific primers (Appendix 2). All amplifications were performed for 35 cycles by use of the following conditions: 94°C for 2 minutes, 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 72°C for 7 minutes with a PCR thermal cyclerm; PCR products were developed after electrophoresis on a 2.0% agarose gel by staining with a proprietary polynucleotide gel stain.i Bands were excised, purified, and submitted for sequence confirmation.
NP-40–soluble material—Approximately 0.35 g of snap-frozen segments of dorsal laminae was pulverized in a prechilled (on dry ice) biopulverizerh and immediately homogenized in 10 mL of extraction buffer (50mM Tris [pH, 7.0], 150mM NaCl, 5mM EDTA, 0.5% NP-40 containing 10μM E64, 1.5μM pepstatin A, and 1mM phenylmethanesulfonyl fluoride) on ice. The homogenized sample was incubated overnight (approx 15 hours) at 4°C and centrifuged (14,000 × g) for 15 minutes at 4°C; supernatant then was collected, and protein in the supernatant was precipitated by the addition of ice-cold absolute ethanol to achieve a final concentration of 80% (vol/vol). The precipitate was washed twice with ice-cold 80% ethanol, dried under a stream of nitrogen gas, and dissolved in PBS solution. Protein concentration was determined via a colorimetric assay based on a protein-binding dye.n
Guanidine hydrochloride-soluble material—Approximately 0.35 g of snap-frozen segments of dorsal laminae was pulverized in a prechilled (on dry ice) biopulverizerh and homogenized in 5 mL of cold extraction buffer (0.1M PBS solution, with 5mM iodoacetic acid, 0.1mM 4-[2-aminoethyl] benzenesulphonyl fluoride, 1% 3-[{cholamidopropyl}-dimethylammonio]-1-propane-sulfonate, 1 μg of pepstatin A/mL, 50mM sodium acetate, 5mM benzamidine hydrochloride hydrate, 5mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, and 4M guanidine hydrochloride; pH, 7.6) supplemented with a proteinase inhibitor cocktailo; samples were homogenized for 30 seconds on ice and extracted overnight at 4°C. The samples were centrifuged at 14,000 × g for 30 minutes at 4°C, a floating layer of insoluble lipids was removed, and the remaining supernatant was collected and precipitated overnight with 4 volumes of ice-cold absolute ethanol containing 5mM sodium acetate. Precipitated molecules were collected via centrifugation at 4°C for 1 hour at 14,000 × g and then dried under a stream of nitrogen gas. The pellet was resuspended in buffer and digested with 0.06 U of Streptomyces hyaluronidasep/10 μg of original frozen tissue as per the manufacturer's instructions. After digestion, the supernatant solids were precipitated by addition of ice-cold absolute ethanol containing 5mM sodium acetate to achieve a final concentration of 80% (vol/vol), dried, and digested with 0.01 U of chondroitinase ABCq/10 μg of original frozen tissue as per the manufacturer's instructions. Supernatant solids were again precipitated, dried, and digested with 10 μU of keratanase IIq/10 μg of original frozen tissue. The digestion protocol allowed solubilization of the many molecules that form insoluble macromolecular complexes with hyaluronan and their subsequent analysis by SDS-PAGE.
SDS-PAGE and western blotting—An aliquot (30 μg of protein content) of extract was boiled in reducing Laemmli (5mM 2-mercaptoethanol) sample bufferr for 5 minutes and subjected to SDS-PAGE in a 4% (wt/vol) polyacrylamide stacking gel with a 10% (wt/vol) polyacrylamide gel as previously described.23 Proteins were transferred to polyvinylidene fluoride membranes via electroblotting. The membranes were blocked by incubation with 5% dry milk in PBS solution with 0.05% Tween-20 for 1 hour, washed with PBS solution with 0.1% Tween-20 for 30 minutes, and then incubated overnight at 4°C with primary antibodies24,s–aa (Appendix 3). Antibodies were used together with immunoaffinity-purified rabbit polyclonal anti-NTPEDSDPDHFD (ADAMTS-4 metalloproteinase domain epitope). After incubation with primary antibodies, the membranes were washed twice in PBS solution with 0.1% Tween-20 for 30 minutes and incubated with secondary antibodies conjugated with HRP. Detection was performed via enhanced chemiluminescencebb and evaluated by use of a gel imaging and documentation system,cc and quantification was performed with associated software.dd
Immunofluorescence—Frozen sections (thickness, 10 μm) were cut from embeddeda tissues and affixed to treated glass slides.ee Immunofluorescent staining was performed in accordance with previously described methods.25 Briefly, slides were blocked by incubation with 5% BSA in PBS solution with 0.001% Tween-20; slides were then treated by incubation for 1 hour at 21°C with optimal dilutions of primary antibodiesff–nn (Appendix 4). Sections were washed and treated by incubation for 1 hour at 21°C with secondary antibodies along with a 1:2,000 dilution of DAPI. Actin was developed by incubating tissue sections for 1 hour at 21°C with a 1:200 dilution of phalloidin-FITCoo conjugate.
Specificity of staining was established via enzyme digestion and peptide blocking. Enzymatic digestions were performed for 1 hour at 37°C. For destruction of hyaluronan epitopes, sections were treated with 0.01 U of chondroitinase ABCoo in 20 μL of 50mM Tris (pH, 6.8) and 60mM sodium acetate with 0.2% BSA. After digestion, sections were thoroughly washed with PBS solution with 0.001% Tween-20 and reacted with antibodies as described previously. For peptide competitions, antibodies were incubated overnight with a 10-fold excess of cognate peptide or antisense peptide in 1% BSA at 4°C prior to incubation with sections as described. All slides were evaluated by use of an inverted microscope with a gridpp; UV, blue, and green excitation light; and 20× or 63× magnification of the objective.pp
Results
Gene expression—Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in the digital laminae of 8 healthy horses as determined via RT-qPCR assays. Primer sets each generated a single product with the expected size (Figure 1) and sequence (data not shown). Mean ± SD cycle threshold values were 21.82 ± 0.88 for GADPH, 29.02 ± 1.37 for aggrecan, 25.84 ± 1.20 for versican, 30.34 ± 0.94 for hyaluronan synthase II, 34.12 ± 1.57 for ADAMTS-4 (N-terminal domain primers), and 32.45 ± 1.05 for ADAMTS-4 (C-terminal domain primers). No cycle threshold value was recorded for the blank control sample.
Agarose gel of RT-qPCR assay products generated by use of primer pairs specific for aggrecan, versican, hyaluronan synthase II, and ADAMTS4 and cDNA generated by use of RNA from healthy laminae of 8 horses. Lanes are as follows: 1, DNA marker (100-bp ladder); 2, aggrecan; 3, versican; 4, hyaluronan synthase II; 5, ADAMTS-4 N-terminal domain; 6, ADAMTS-4 C-terminal domain; 7, GAPDH; and 8, blank control sample. Numbers on the left side represent the number of base pairs.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1035
Comparative RT-qPCR analyses performed with primer sets specific for equine versican G1, αGAG, βGAG, and C-terminal domains revealed ratios of approximately 1:1:1:1 (data not shown), which is consistent with expression of the full-length gene. In addition, RT-PCR analyses performed with primer sets specific for equine isoform V0 αGAG and βGAG (Figure 2), isoform V1 G1 and βGAG, isoform V2 G1 and αGAG, and isoform V3 G1 and C revealed that spliced sequences encoding V0 and V1 isoforms were abundantly present in laminae, whereas those encoding the V2 isoform were less abundant, and spliced sequences encoding the V3 isoform were not detected. Primer specificity was confirmed by sequencing the products (data not shown).
Agarose gel of PCR assay products generated by use of primer pairs specific for versican isoforms and cDNA generated by use of RNA from healthy laminae of 8 horses. Lanes are as follows: 1, DNA marker (100-bp ladder); 2, isoform V0; 3, isoform V1; 4, isoform V2; and 5, isoform V3. Numbers on the left side represent the number of base pairs.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1035
Protein expression—Extracts from the 8 laminae were subjected to SDS-PAGE and western blotting. Protein expression of ADAMTS-4 and its substrates was detected (Figure 3).
Western blots of the immunoreactivity of ADAMTS-4 and its substrates in extracts of healthy laminae of 8 horses. Laminar extracts (30 μg of protein/lane) were probed with antibody against neoepitope FASLSRFVET exposed on ADAMTS-4 catalytic domain after removal of the regulatory propeptide (NP-40 extract; A); antibody against V0 and V1 neoepitope DPEAAE generated by ADAMTS-4 cleavage (NP-40 extract; B); antibody against V0 and V2 neoepitope NIVSFE generated by ADAMTS-4 cleavage (NP-40 extract; C); and antibody against aggrecan neoepitope ARGSVIL (BC-3) generated by ADAMTS-4 cleavage (guanidine hydrochloride extract digested with hyaluronidase, chondroitinase ABC, and keratanase III; D). Numbers to the left of each column represent the number of kilodaltons.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1035
ADAMTS-4
Proteins in NP-40 extract of laminae were subjected to SDS-PAGE, and ADAMTS-4 was detected with western blotting by means of an antibody specific for the catalytic site cleavage neoepitope FASLSRFVET, which is revealed only on removal of the regulatory propeptide.24 Results indicated that processed ADAMTS-4 was predominantly present as an approximately 51-kDa polypeptide accompanied by an approximately 41-kDa polypeptide (Figure 3). The approximately 51-kDa polypeptide was detected in all 8 laminae extracts, whereas the approximately 41-kDa polypeptide was detected in only 6 of 8 laminae extracts. The same results were obtained when samples of pulverized laminae were directly solubilized in SDS–sample buffer (data not shown); this indicated that processing of ADAMTS-4 by propeptide convertase,26,27 which exposes the FASLSRFVET catalytic site neoepitope, was not an extraction artifact.
V0 and V1 isoforms
To explore versican protein expression, NP-40 laminae extracts were subjected to SDS-PAGE, and ADAMTS-4 V0 and V1 cleavage fragments were revealed via western blotting by means of an antibody specific for the C-terminal V0 and V1 βGAG domain neoepitope DPEAAE. Results revealed V0 and V1 fragments of approximately 109 and 66 kDa, respectively (Figure 3). Detection of the polypeptides was abrogated when anti-DPEAAE antibodies were preincubated with the sense peptide but not the antisense peptide (data not shown). The approximately 109-kDa polypeptide was detected in 6 of 8 laminae extracts, whereas the approximately 66-kDa polypeptide was detected in all 8 laminae extracts. Polypeptide bands of similar size were detected when pulverized laminae were directly solubilized in SDS-sample buffer (data not shown), which indicated that processing of V0 and V1 isoforms by ADAMTS-4 was not an extraction artifact.
V0 and V2 isoforms
The NP-40 laminae extracts were subjected to SDS-PAGE, and ADAMTS-4 V0 and V2 cleavage fragments were revealed via western blotting by means of an antibody specific for the C-terminal V0 and V2 αGAG neoepitope NIVSFE. Results revealed V0 and V2 fragments of approximately 112 and 68 kDa, respectively (Figure 3). The polypeptides were detected in all 8 laminae extracts, and detection of bands was abrogated when antibodies were preincubated with the sense peptide but not the antisense peptide (data not shown). Polypeptide bands of similar size were detected when tissue was directly solubilized in SDS—sample buffer (data not shown), which indicated that processing of V0 and V2 isoforms by ADAMTS-4 was not an extraction artifact.
Aggrecan
The SDS-PAGE analysis of ADAMTS-4 fragments of aggrecan required extraction of the fragments from pulverized laminae by means of guanidine hydrochloride buffer, precipitation with ethanol, and subsequent digestion with hyaluronidase, chondroitinase ABC, and keratanase (data not shown). These treatments were necessary because aggrecan ADAMTS-4 cleavage fragments were not extracted from pulverized laminae via incubation with NP-40 buffer, were coextracted with their binding partner hyaluronan in guanidine hydrochloride buffer, were held in an insoluble hyaluronan gel on replacement of guanidine hydrochloride with water or physiologic buffer, and were heavily glycosylated (particularly with chondroitin sulfate GAGs), which prevented migration through the SDS-PAGE stacking gel in the absence of appropriate digestion. Western blotting performed following SDS-PAGE of the hyaluronidase-, chondroitinase ABC–, and keratanase-digested guanidine hydrochloride–extracted material by use of an antibody specific for the aggrecan N-terminal ADAMTS-4 cleavage neoepitope ARGSVIL revealed bands at approximately 318, 250, 150, and 70 kDa (Figure 3). These polypeptide bands were detected in all 8 laminae extracts.
Immunohistologic evaluation—Immunohistologic evaluations were performed on flash-frozen embedded laminae (n = 6) to determine the cellular localization of ADAMTS-4 and its substrates. All 6 samples were analyzed for each staining protocol.
Basement membrane
Thin transverse sections of frozen digital laminae were stained with a pan–laminin-specific antibody, Texas red–conjugated secondary antibody, and the DNA-intercalating dye (ie, DAPI; Figure 4). Laminin is a component of the basement membrane that is located at the junction between the secondary epidermal and dermal laminae. Laminin is also a component of the basement membrane of small blood vessels, which were visible in the secondary dermal laminae, and of blood vessels in the primary dermal laminae. Throughout the tissue, cell nuclei stained with DAPI were visible, and those of the basal epithelial cells abutted the basement membrane. Some tissue components, putatively collagen fibers in primary and secondary dermal laminae, autofluoresced at the excitation wavelength (488 nm) used in the study.
Photomicrographs of immunohistochemically stained sections of equine laminae reveal the lamellar structure and epidermal-dermal boundaries via localization of laminin and actin. A—The epidermal-dermal lamellar boundary is defined by immunofluorescent staining against the basement membrane marker laminin (red). B—Epidermal cellular boundaries are defined by immunofluorescent staining against actin (green). Autofluorescent material (putatively collagen) is green, and nuclei are blue. Bars = 50 μm.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1035
Cortical actin
Thin transverse sections of frozen digital laminae were stained with phalloidin-FITC to detect actin bundles and with DAPI to detect DNA in nuclei (Figure 4). Phalloidin-FITC was most densely associated with the cortical actin skeleton of basal epithelial cells (the outermost cell layer of the secondary epidermal laminae) but also defined the cortical actin skeleton of suprabasal cells extending into the primary epidermal laminae. Nuclei stained with DAPI were faded or not visible in keratinocytes in the primary epidermal laminae. The contiguous keratinocyte cortical actin network was evident throughout the secondary epidermal laminae, which indicates that there was little room for ECM in this tissue. The phalloidin-FITC stain was bright, and little exposure time was required to obtain images, which accounted for the almost complete lack of green autofluorescent materials.
Versican
Thin transverse sections of frozen digital laminae were stained with antibody specific for the carboxy-terminal domain of all versican isoforms, a secondary antibody conjugated with a proprietary red dye, and DAPI (Figure 5). Staining for versican was restricted to a single layer of cells, putatively the basal epithelial cells. Specific staining for versican was inhibited by preincubation of the antibody with its competing peptide but not with an antisense peptide (data not shown).
Photomicrographs of immunohistochemically stained sections of equine laminae reveal that versican uniquely localizes to the basal epithelia of the secondary epidermal lamellae and is not associated with the basement membrane. A—Versican (red) is evident via immunofluorescent staining. B—Versican staining is blocked by preincubation of primary antibody with cognate peptide. C—Versican (red) and the basement membrane are evident by staining against laminin (green). D—Epithelial cell boundaries are defined by staining against actin (green). Bars = 50 μm. See Figure 4 for remainder of key.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1035
Identification of the versican-stained cell layer as basal epithelial cells was confirmed on the basis that the cells abutted the basement membrane. This was verified by staining a thin section of frozen laminae with pan–anti-laminin antibody (which was revealed by use of a secondary antibody conjugated with a proprietary green fluorescent dye), anti-versican antibody (which was revealed by use of red fluorescent dye–conjugated secondary antibody), and DAPI (which stained the nuclei; Figure 5). The versican did not actually contact (colocalize with) the basement membrane laminin, which would have yielded an image with merged colors. To test whether versican was contained within basal epithelial cells, a thin cryosection was stained with anti-versican antibody, which was revealed by use of Texas red–conjugated secondary antibody, phalloidin-FITC to detect actin bundles, and DAPI to detect cell nuclei. Results indicated that versican was located between the cortical actin of basal epithelial cells and the cell nuclei. In addition, versican staining was not pronounced along cell boundaries, which suggested little or no accumulation of the proteoglycan in the extracellular space.
Aggrecan
Thin transverse sections of frozen digital laminae were stained with antibody that reacted with an epitope on the G2 domain of aggrecan, proprietary red fluorescent dye–conjugated secondary antibody, and DAPI (Figure 6). Aggrecan staining was greatest in basal epithelial cells but was also detected in regions of the epidermal laminae occupied by suprabasal epithelial cells. Aggrecan was largely, if not entirely, absent from primary and secondary dermal laminae. Autofluorescent material (putatively collagen) was readily detected. Aggrecan-staining was inhibited by preincubation of the antibody with its competing peptide. To further identify the distribution of aggrecan in laminae, a transverse thin frozen section was stained with aggrecan specific antibody, phalloidin-FITC to detect actin bundles, and DAPI to detect nuclei. Aggrecan staining was evident within punctuate bodies contained within boundaries defined by phalloidin-stained cortical actin. A similar tissue distribution was seen in immunohistologic sections stained with the antibody against the ARGSVIL epitope of aggrecan (data not shown).
Photomicrographs of immunohistochemically stained sections of equine laminae reveal that aggrecan localizes primarily to the secondary epidermal lamellae. A—Aggrecan (red) is visible. Bar = 50 μm. B—Aggrecan staining is blocked by preincubation of the primary antibody with cognate peptide. Bar = 20 μm. C—Aggrecan (red) and epithelial cell boundaries are evident by staining against actin (green). Bar = 20 μm. See Figure 4 for remainder of key.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1035
Hyaluronan
Thin transverse sections of frozen digital laminae were stained with antibody specific for hyaluronan polysaccharide, Texas red–conjugated secondary antibody, and DAPI. Hyaluronan was diffusely present in the epidermal laminae and not pronounced along cell boundaries (Figure 7). Staining was not detected in tissue sections that had been pretreated with hyaluronidase. Hyaluronan staining within the secondary epidermal laminae was contained within cell boundaries defined by phalloidin-stained cortical actin.
Photomicrographs of immunohistochemically stained sections of equine laminae reveal that hyaluronan is present throughout the lamellae but enriched in the secondary epidermal lamellae. A—Hyaluronan (red) is visible. Bar = 50 μm. B—Hyaluronan staining is abrogated by incubation of tissue section with an epitope-digesting enzyme (chondroitinase ABC [pH, 6.8]). Bar = 20 μm. C—Hyaluronan (red) and epithelial cell boundaries are evident by staining against actin (green). Bar = 20 μm. See Figure 4 for remainder of key.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1035
ADAMTS-4
Thin transverse sections of frozen digital laminae were stained with an antibody specific to the peptide NTPEDSDPDHFD, which corresponded to amino acids 300 to 311 of the metalloprotease domain of equine ADAMTS-4. Staining of ADAMTS-4 was performed with a red fluorescent dye–conjugated secondary antibody, and nuclei were counterstained with DAPI. Antibody specificity was confirmed via western blotting, which revealed band patterns similar to those obtained by use of the FASLSRFVET ADAMTS-4 antibody (Figure 3) and via inhibition of western blotting with the sense but not the antisense peptide (data not shown).
Staining was diffusely present throughout the epithelial cells of the secondary epidermal lamellae (Figure 8). It was abrogated by preincubation of the antibody with its immunizing peptide but not with a noncompeting peptide (data not shown). Punctate staining was also evident in cells associated with the vasculature, mononuclear cells of the dermal lamellae, and dermal fibroblasts.
Photomicrographs of immunohistochemically stained sections of equine laminae reveal that ADAMTS-4 is present in epithelial cells of the secondary epidermal lamellae. A—Notice that there is staining of the ADAMTS-4 NTPEDSDPDHFD epitope (red). Bar = 50 μm. B—Staining of the ADAMTS-4 NTPEDSDPDHFD epitope is blocked by cognate peptide. Bar = 20 μm. C—A higher-magnification image reveals ADAMTS-4 (red), vascular endothelia (white arrows), mononuclear cells of the dermal lamellae (blue arrows), and a dermal fibroblast (yellow arrow). Bar = 50 μm. See Figure 4 for remainder of key.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1035
Discussion
In the study reported here, RT-qPCR assays and validated primers were used to determine that equine digital laminae express genes encoding ADAMTS-4, aggrecan, versican (processed to V0, V1, and V2 isoforms), and hyaluronan synthase II. By use of SDS-PAGE followed by western blotting with antibodies specific for conserved peptides in equine and human ADAMTS-4, aggrecan, and versican and validation by peptide competition, we confirmed that gene expression was accompanied by protein expression and that the core proteins of the large polysulfated proteoglycans were subject to constitutive cleavage by ADAMTS-4 in vivo. Through immunofluorescent and immunohistochemical staining of thin sections of laminae with specific antibodies validated by peptide competition or targeted epitope digestion, we found that aggrecan, versican, and hyaluronan were predominantly present within basal epithelial cells of the secondary epidermal laminae, whereas ADAMTS-4 was present within these cells and also in cells of the dermal laminae.
In all laminar extracts, ADAMTS-4 was present predominantly as an approximately 51-kDa form bearing the conserved FASLSRFVET neoepitope, which is exposed on removal of the regulatory propeptide by furin propeptide convertase in the trans golgi.26 A minor, approximately 61-kDa form bearing the neoepitope was also detected in all samples, but staining was too weak to be visible in photomicrographs. In addition, an approximately 42-kDa form of ADAMTS-4 bearing the FASLSRFVET neoepitope was detected in extracts from several samples. Processed ADAMTS-4 bearing the FASLSRFVET neoepitope has a molecular weight between approximately 68 and 70 kDa in mice and humans, and a portion of the material undergoes autoproteolysis to generate polypeptides of approximately 61, 51, and 40 kDa,19,28,29 which corresponded to the approximately 61-, 51-, and 42-kDa laminae material reported here. The absence of the approximately 68- to 70-kDa enzyme from extracts of laminae suggested that autoproteolytic activity of equine ADAMTS-4 may be greater than that of murine and human ADAMTS-4.
In addition to cleaving the large polysulfated proteoglycans, autoproteolyzed ADAMTS-4 can cleave low–molecular weight, leucine-rich proteoglycans (which bind collagens and fibronectin), thereby affecting fiber formation and network organization.29,30 Thus, in equine digital laminae, ADAMTS-4 may cause reorganization of a broad range of ECM components. This increased cleavage capability may have implications for the development of laminitis.
Aggrecan is a highly glycosylated multidomain protein.18 The core protein of approximately 220 kDa can contain up to 100 chondroitin sulfate side chains, each of approximately 20 kDa and positioned between globular domains 2 and 3. Aggrecan can also contain up to 60 keratin sulfate side chains, each of approximately 5 to 15 kDa, which are more widely distributed on the molecule than are the chondroitin sulfate GAGs. In addition, a variable number of O- and N-linked oligosaccharide side chains can also be added. Cleavage of aggrecan between the G1 and G2 domains at the Glu373-Ala374 bond by ADAMTS-4 yields a large GAG-rich fragment with an N-terminal ARGSVI cleavage neoepitope and a G1 fragment with a NITEGE neoepitope. Antibodies specific for ARGSVIL revealed polypeptides of approximately 318, 250, 150, and 70 kDa in guanidine hydrochloride extracts of laminae that were further subjected to digestion with hyaluronidase–chondroitinase ABC. The largest fragments most likely corresponded to N- or O-glycosylated material. The presence of polypeptides bearing the ADAMTS-4 cleavage neoepitope in lamina extracts indicated that endogenous ADAMTS-4 was active in the laminae.
Cleavage of aggrecan between Glu373-Ala374 by ADAMTS-4 and ADAMTS-5 in cartilage allows large GAG-rich fragments, which are no longer anchored to hyaluronan, to diffuse into synovial fluid.31 However, constitutive cleavage of aggrecan in the laminae did not allow the ARGSVI-positive GAG-rich fragments to easily detach from the tissue, which was inferred from our inability to extract the fragments with NP-40 homogenization buffer. Rather, overnight incubation of pulverized laminae in a 4M guanidine hydrochloride extraction buffer was required to extract the cleavage fragments, which suggested that they were held in the lamellar tissue by interactions with other unidentified molecules.
The NP-40 and direct SDS–sample buffer extracts of pulverized laminae contained V0 and V1 fragments of approximately 109 and 66 kDa, respectively, bearing the C-terminal βGAG domain ADAMTS-4 cleavage neoepitope DPEAAE. The molecular weights of these fragments were unaffected by digestion with hyaluronidase or chondroitinase ABC; hence, the fragments did not have attached hyaluronan or chondroitin sulfate GAGs. It is likely that the approximately 66-kDa fragment described in the present report is equivalent to the approximately 70-kDA DPEAAE–positive G1 domain of V1 reported in ADAMTS-4–treated human aorta.32 The extracts of laminae also contained approximately 112- and 68-kDa V0 and V2 fragments bearing the C-terminal ADAMTS-4 αGAG domain cleavage neoepitope NIVSFE. Cleavage of fully deglycosylated human aorta V0 and V2 by ADAMTS-4 has been found to yield an NIVSFE-positive fragment of approximately 64 kDa, which is equivalent to the glial hyaluronate–binding protein of human brain V2.33 Although we digested extracts of laminae with chondroitinase ABC, we did not digest them with sialidase or O-glycanase, which perhaps accounted for the larger-size (approx 68-kDa) putative versican G1-αGAG domain fragment detected in the present study. However, the approximately 112-kDa material was too large to be comprised solely of the ADAMTS-4–cleaved versican G1 and αGAG core protein domains, which suggested that it may be heavily N-glycosylated. The approximately 112- and 68-kDa polypeptides bearing the NIVSFE neoepitope were also detected (data not shown) by antibodies raised against the N-terminal 13-residue peptide sequence of human versican (LF-9934,qq), which is 87% homologous to the matched equine sequence; this further supported their identity as fragments of versican.
Aggrecan and versican have been extensively studied in articular cartilage, tendon, and atherosclerotic plaque, where they associate with hyaluronan in the ECM.12,18,35 Hyaluronan is a nonsulfated GAG that is synthesized at the plasma membrane by 1 of 3 hyaluronan synthases36 and extruded into the extracellular space. Hyaluronan synthase II, which we found to be expressed in the laminae, makes the longest hyaluronan chains, which can be > 2,000 kDa. Hyaluronan binds a large number of aggrecan monomers, with binding stabilized by link protein. These complexes can be several hundred million daltons.13 The large, negatively charged complexes of aggrecan and hyaluronan attract and hold water in tissues, forming a hydrated gel. This is packaged in collagen fibers in articular cartilage and tendon, which accounts for the high resistance of these tissues to compression deformation.18 Versican is also a hyaluronan-binding proteoglycan. It is expressed in fast-growing cells of many tissues, including the skin and the media of the aorta and in developing limb buds of chickens,37 where it is implicated in regulating cell proliferation and migration as well as in expanding the ECM and increasing its viscoelasticity. Thus it was expected that aggrecan, versican, and hyaluronan in the digital laminae would also be associated with ECM and present in producer cells. However, the materials were not detected in the ECM. Furthermore, the secondary epidermal laminae had little or no discernible ECM.
Aggrecan was detected in punctuate bodies contained within cortical actin boundaries throughout the basal and suprabasal epithelial cells of the secondary epidermal laminae. Hyaluronan staining was also detected in punctuate bodies contained within cortical actin boundaries throughout the secondary epidermal laminae. In contrast, versican staining was restricted to only basal epithelial cells. Versican was not detected in any suprabasal epithelial cells in the secondary epidermal laminae. Thus, results of the present study suggest that versican, aggrecan, and hyaluronan may be useful markers for defining specialization within epidermal keratinocytes.
In addition to aggrecan, versican, and hyaluronan, basal epithelial cells also contained ADAMTS-4. Because versican fragments bearing an ADAMTS-4 neoepitope were readily extracted from laminae into SDS–sample buffer, which prevents postextraction processing, it can be concluded that a portion (if not all) of versican and ADAMTS-4 shared an intracellular compartment that permitted ADAMTS-4 activity. Furthermore, because aggrecan fragments bearing an ADAMTS-4 neoepitope could also be extracted from laminae, it is likely that at least a portion of aggrecan and ADAMTS-4 also shared an intracellular compartment that permitted ADAMTS-4 activity. Thus, ADAMTS-4 contributed to processing and, most likely, turnover of aggrecan and versican within laminar basal epithelial cells.
The biological roles of aggrecan, versican, hyaluronan, and ADAMTS-4 in equine digital laminae were not directly evaluated in the present study. However, on the basis of known compression-resistance and cell-signaling properties of the large polysulfated proteoglycans, it is reasonable to propose that the proteoglycans affect development and maintenance of the basal epithelial cell layer and may cushion basal epithelial cells against severe biomechanical stresses associated with their anatomic location.
ABBREVIATIONS
| ADAMTS-4 | A disintegrin and metalloproteinase with thrombospondin motifs-4 |
| BSA | Bovine serum albumin |
| DAPI | 4′,6-diamidino-2-phenylindole |
| ECM | Extracellular matrix |
| FITC | Fluorescein isothiocyanate |
| GAG | Glycosaminoglycan |
| GAPDH | Glyceraldehyde 3-phosphate dehydrogenase |
| HRP | Horseradish peroxidase |
| KLH | Keyhole limpet hemocyanin |
| NP-40 | Tergitol-type nonyl phenoxylpolyethoxylethanol |
| qPCR | Quantitative PCR |
| RT | Real time |
Tissue-Tek OCT, Sakura Finetek USA Inc, Torrance, Calif.
Freund's adjuvant, complete, Sigma-Aldrich, St Louis, Mo.
GenScript USA Inc, Piscataway, NJ.
Freund's adjuvant, incomplete, Sigma-Aldrich, St Louis, Mo.
NHS-activated Sepharose Fast Flow beads, GE Healthcare Biosciences, Pittsburgh, Pa.
NanoDrop 1000, Thermo Scientific, Wilmington, Del.
Stratagene Absolutely RNA kit, Stratagene, La Jolla, Calif.
Biospec Products Inc, Bartlesville, Okla.
SYBRSafe DNA gel stain, Molecular Probes, Eugene, Ore.
Quanta q Script cDNA synthesis kit, Quanta BioSciences, Gaithersburg, Md.
SYBR premix Ex Taq, Applied Biosystems, Foster City, Calif.
Stratagene MX 3005p, Stratagene, La Jolla, Calif.
PTC-100PCR System, MJ Research Inc, Waltham, Mass.
Bradford assay, Bio Rad Life Sciences, Hercules, Calif.
SigmaFast protease inhibitor cocktail, Sigma-Aldrich, St Louis, Mo.
Calbiochem, EMD, Merck KGaA, Darmstadt, Germany.
Seikagaku, Tokyo, Japan.
Laemmli reducing sample buffer, Bio Rad Life Sciences, Hercules, Calif.
No. ab3773, AbCam, Cambridge, Mass.
No. ab6808, AbCam, Cambridge, Mass.
No. ab19345, AbCam, Cambridge, Mass.
No. ab6795, AbCam, Cambridge, Mass.
Antibody provided by Drs. M. D. Tortorella and Dr. A. M. Malfait, Pfizer Global Research and Development, St Louis, Mo.
No. ab8226, AbCam, Cambridge, Mass.
No. ab28671, AbCam, Cambridge, Mass.
No. AB1032, Millipore, Billerica, Mass.
No. AB1033, Millipore, Billerica, Mass.
ECL, Bio Rad Life Sciences, Hercules, Calif.
G: Box, Syngene, Frederick, Md.
Gene Tools, Syngene, Frederick, Md.
Fisher Superfrost Plus, Fisher Scientific, Fair Lawn, NJ.
No. ab11575, AbCam, Cambridge, Mass.
No. ab6719, AbCam, Cambridge, Mass.
No. 711-485-152, Jackson ImmunoResearch Laboratories Inc, West Grove, Pa.
No. 26706, Santa Cruz Biotechnologies, Santa Cruz, Calif.
No. 705-295-147, Jackson ImmunoResearch Laboratories Inc, West Grove, Pa.
No. ab16320, AbCam, Cambridge, Mass.
No. 711-515-152, Jackson ImmunoResearch Laboratories Inc, West Grove, Pa.
No. ab53842, AbCam, Cambridge, Mass.
No. ab6745, AbCam, Cambridge, Mass.
Sigma-Aldrich, St Louis, Mo.
Zeiss MOT200 with Zeiss apotome, Carl Zeiss MicroImaging Inc, Thornwood, NY.
Anti-serum provided by Dr. Larry Fisher, National Institutes of Health, Bethesda, Md.
Appendix 1
Primer sequences used in RT qPCR assay evaluation of gene expression in healthy forelimb laminae obtained from 8 horses.
| Gene | Sequence | GenBank accession No. | Amplicon length (bp) | Primer efficiency (%) | R2 |
|---|---|---|---|---|---|
| ADAMTS-4 (C-terminal) | F: 5′-gctgggctactattatgtgctg-3′ | ||||
| R: 5′-ccacattgttgtatccgtacct-3′ (N-terminal) | 100033914 | 227 | 104 | 0.976 | |
| ADAMTS-4 | F: 5′-cagtatcgaggggaccgaact-3′ | ||||
| R: 5′-gaaatgctgccatcttgtcat-3′ | 100033914 | 241 | 92.6 | 1.000 | |
| Aggrecan | F: 5′-caacaacaatgcccaagactac-3′ | ||||
| R: 5′′-agttctcaaattgcaaggagtg-3′ | 100033876 | 110 | 102 | 0.981 | |
| Versican | F: 5′-cctgcaattaccatctcaccta-3′ | ||||
| R: 5′-cagggagttgatttcataacga-3′ | 100065275 | 122 | 92.1 | 0.988 | |
| Hyaluronan synthase 2 | F: 5′-cacagacaggctgaggacaa-3′ | ||||
| R: 5′-acaggctttggatgatgagg-3′ | 100009708 | 231 | 94 | 0.930 | |
Appendix 2
Primer sequences used for PCR detection of specific versican isoforms in healthy forelimb laminae obtained from 8 horses.
| Primer | Sequence* | Isoform | Primer pair | Expected product size (bp) |
|---|---|---|---|---|
| eqV-F1 | 5′-gctgaagaagagtgtgaaaa-3′ | V0 | eqV-F2 + eqVR-1 | 530 |
| eqV-F2 | 5′-tggtgaagaaacaaccagtg-3′ | V1 | eqV-F1 + eqV-R1 | 520 |
| eqV-F3 | 5′-ctcgtgttcctcccactacc-3′ | V2 | eqV-F3 + eqV-R2 | 510 |
| eqV-R1 | 5′-agtggtgactagatgtttcc-3′ | V3 | eqV-F1 + eqV-R2 | 547 |
| eqV-R2 | 5′-tgggcaaagtacttgtagca-3′ | NA | NA | NA |
GenBank accession No. 100065275.
NA = Not applicable.
Appendix 3
Antibodies used for western blot analysis of protein expression in healthy forelimb laminae obtained from 8 horses.
| Primary antibody | Dilution | Secondary antibody |
|---|---|---|
| Mouse monoclonal (BC-3) to human aggrecan ARGSVILs | 1:2,000 | Sheep polyclonal to mouse IgG H&L (HRP)t |
| Rabbit polyclonal to versican (V0 and V1) DPEAAE (within β domain)u | 1:2,000 | Sheep polyclonal to rabbit IgG H&L (HRP)v |
| Rabbit polyclonal to human ADAMTS-4 catalytic neoepitope FASLSRFVET24,w | 1:500 | Sheep polyclonal to rabbit IgG H&L (HRP)v |
| Mouse monoclonal to β-actin–loading controlx | 1:20,000 | Sheep polyclonal to mouse IgG H&L (HRP)t |
| Rabbit polyclonal to versican (V0 and V2) NIVSFE (within α domain)y | 1:2,000 | Sheep polyclonal to rabbit IgG H&L (HRP)v |
| Rabbit polyclonal to mouse versican (αGAG domain) amino acids 535 to 598 of mouse versican V0 and V2z | 1:1,000 | Sheep polyclonal to rabbit IgG H&L (HRP)v |
| Rabbit polyclonal to mouse versican (β GAG domain) amino acids 1,360 to 1,439 of mouse versican V1aa | 1:1,000 | Sheep polyclonal to rabbit IgG H&L (HRP)v |
IgG H&L = Heavy and light chains of immunoglobulin of the IgG class.
Appendix 4
Antibodies used for immunofluorescent localization in heathy forelimb laminae obtained from 8 horses.
| Primary antibody | Dilution | Secondary antibody |
|---|---|---|
| Rabbit polyclonal to lamininff | 1:100 | Goat polyclonal to rabbit IgG H&L Texas red conjugategg |
| Donkey polyclonal to rabbit IgG H&L conjugated with a flurochrome with mean ± SD excitation and emission* of 493 ± 4 nm and 518 ± 4 nm, respectivelyhh | ||
| Goat polyclonal to human versican (T-20)ii | 1:20 | Donkey polyclonal to goat IgG H&L conjugated with a fluorochrome with mean ± SD excitation and emission* of 591 ± 4 nm and 616 ± 4 nm, respectivelyjj |
| Rabbit polyclonal to human aggrecan G2 domain peptide (amino acids 89 to 106)kk | 1:50 | Donkey polyclonal to rabbit IgG H&L conjugated with a flurochrome with mean ± SD excitation and emission* of 591 ± 4 nm and 616 ± 4 nm, respectivelyll |
| Sheep polyclonal to human hyaluronic acidmm | 1:250 | Rabbit polyclonal to sheep IgG H&L Texas red conjugatenn |
| Affinity-purified rabbit polyclonal to equine ADAMTS-4 metalloprotease domain peptide (amino acids 300 to 311) | 1:20† | Donkey polyclonal to rabbit IgG H&L conjugated with a fluorochrome with mean ± SD excitation and emission* of 591 ± 4 nm and 616 ± 4 nm, respectivelyll |
Excitation and emission was in PBS solution.
Generated as part of the present study.
IgG H&L = Heavy and lightchains of immunoglobulin of the IgG class.
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