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DOI: 10.1111/j.1365-3164.2008.00732.xBlackwellPublishingLtd

Increased elastic microbrils and thickening ofbroblastic nuclear lamina in canine cutaneous asthenia

M. H. Bellini* ,, E. T. E. G. Caldini,M. P. Scapinelli, M. J. Simes,D. B. Machado* and R. Nrmberg

,

*Energy and Nuclear Research Institute, IPEN-CNEN/SPUniversity of So Paulo, USPFaculdades Metropolitanas Unidas, UNIFMUFederal University of So Paulo, UNIFESP-EPMCorrespondence: Dr Maria Helena Bellini, PhD, IPEN-CNEN-SP,Av. Dr Lineu Prestes n. 2242, So Paulo 055080-000, Brazil.

E-mail: mhmarumo@terra.com.br

Sources of Funding

This study was supported by FAPESP.

Conict of Interest

No conict of interest has been declared.

Abstract

Cutaneous asthenia is a hereditary connective tissuedisease, primarily of dogs and cats, resembling EhlersDanlos syndrome in man. Collagen dysplasia results inskin hyperextensibility, skin and vessel fragility, andpoor wound healing. The purpose of this study was todescribe the histological ndings in a dog with acollagenopathy consistent with cutaneous asthenia.An 8-month-old crossbreed female dog presentedwith lacerations and numerous atrophic and irregularscars. The skin was hyperextensible and easily torn bythe slightest trauma. Ultrastructurally, the dermis wascomprised of elaunin and oxytalan microbrils. Theseare immature bres in which the brillar componentis increased but elastin is reduced. Collagen breswere profoundly disorganized. The brils had a highlyirregular outline and a corroded appearance whenviewed in cross-section, and were spiralled andfragmented in a longitudinal view. Dermal broblastsdisplayed a conspicuous thickening of the nuclearlamina. Nuclear lamins form a brous nucleoskeletalnetwork of intermediate-sized laments underlyingthe inner nuclear membrane. Mutations in lamins orlamin-associated proteins cause a myriad of geneticdiseases collectively called laminopathies. Disruptionof the nuclear lamina seems to affect chromatinorganization and transcriptional regulation ofgene expression. A common link among all laminopa-thies may be a failure of stem cells to regeneratemesenchymal tissue. This could contribute to theconnective tissue dysplasia seen in cutaneousasthenia.

Accepted 14 October 2008

Introduction

EhlersDanlos syndrome (EDS) comprises a group ofheterogeneous connective tissue disorders recognizedprimarily by skin hyperextensibility, joint hypermobility andtissue fragility. They display remarkable genetic heterogeneityand can be caused by mutations in several different genesthat encode extracellular matrix (ECM) proteins and theirprocessing enzymes. In humans, EDS are divided into sixmain categories, although clinical phenotypes frequently

overlap.

1

Genetic testing is currently limited by the varietyof molecular defects in the different types of EDS and bythe fact that no major gene has, as yet, been identied forthe most common EDS, the hypermobility type. Mutationsin the genes encoding the

1(I),

2(I),

1(III),

1(V) and

2(V) procollagen chains, lysyl hydroxylase and tenascin-Xhave been found in different forms of EDS and the relatedanimal disease dermatosparaxis, a recessively inheritedconnective tissue disorder.

1,2

EDS type VIIC results froma defect in the processing of type I procollagen to collagenwith accumulation in most tissues of molecules that retainthe amino-terminal propeptide.

3

EDS is also seen in many animal species, including

horses, cats, dogs and rabbits.

48

In dogs, it is mostfrequently observed in dachshunds, boxers, St Bernards,German shepherd dogs, Springer spaniels, greyhounds, Irishsetters and poodles, and is known as cutaneous asthenia.

9

Clinical symptoms are mostly conned to the integument.The skin of affected animals is thin and hyperextensibleand prone to injuries, which typical results in mildlyhaemorrhagic wounds, atrophic scars and occasionallyhaematomas.

10

Histologically, collagen bres show altera-tions in arrangement, length and staining characteristics.

4

Electron microscopy has revealed irregular patterns andvariation in the cross-section diameter of the bres.

11

Both dominant and recessive forms of inheritance have

been described in cats. Initially only a dominant form wasdescribed in dogs, but a recessive type has recently beenpostulated.

10

Here, we describe the histopathological and ultrastructuralndings of a case of cutaneous asthenia in a dog associatedwith novel alterations in the broblast nuclear lamina.

Case report

An 8-month-old crossbred female dog (Doberman

Lab-rador retriever) presented in a poor general condition withskin lacerations due to minor injuries, poor wound healingand lameness of the left hind limb. Numerous atrophic and

irregular scars were found on the more exposed areas ofthe legs. Skeletal development was normal. The ownerreported that the dog had healthy parents and that the

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clinical signs were rst observed when the animal wasapproximately 2 months old.

Skin biopsies of the dorsum and legs from the affectedand from a control dog were examined by light microscopyto conrm the presence of a collagen defect. For lightmicroscopy, samples were xed in neutral 10% bufferedformalin and embedded in parafn. Sections 45

m thickwere stained with haematoxylin and eosin, Massonstrichrome for collagen bres, and Weigerts resorcinfuchsinstain with previous oxidation using 1% oxone to revealoxytalan, elaunin and mature elastic bres.

12

The imageswere captured with a digital camera (DXM1200F; Nikon

Instruments Inc., Melville, NY, USA) and analysed usingthe EclipseNet software for Nikon cameras.Small skin fragments were xed in 2% glutaraldehyde

dissolved in 0.5M

phosphate buffer at pH 7.2, followed bypostxation in 1% osmium tetraxide for 1 h and overnightblock staining in 0.5% aqueous uranyl acetate. The tissueswere embedded in a polyester resin (Polylite, ReichholdResearch Triangle Park, NC, USA), thin-sectioned in an LKBUltratome microtome, double-stained by uranyl acetateand lead citrate, and studied under a Zeiss EM 9S electronmicroscope.

Histopathologically, the thickness of the epidermis anddermis was not altered compared to healthy controls.

Collagen bres, however, were decreased in number,disordered, fragmented and shortened (Figs 1 and 2).There was also a low number of elastic bres, which lacked

the normal appearance and organization of the dermalelastic network (Fig. 3). In some areas local haemorrhage,increased vascularization and broblast accumulationwere noted (Fig. 1).

Transmission electron microscopy revealed profounddisorganization of the collagen bres compared to healthycontrol skin. The brils had a highly irregular outline and acorroded appearance when viewed in cross-section, andwere spiralled and fragmented in longitudinal view. Someof the thicker brils seemed to have resulted from a partialfusion of two or more brils. Several broblasts showedthickening of the nuclear lamina (Fig. 4).

Elastic bres were reduced in number and were mostlymicrobrils. Eulanin and oxytalan elastic bres predominatedin the affected dogs dermis, in contrast to healthycontrol skin where mature elastic bres were moreabundant (Fig. 5).

Discussion

This report describes the histopathological ndings in acase of cutaneous asthenia in a dog. Light microscopyclearly showed morphological changes in the collagen.These alterations have been also been described in earlierreports of canine cutaneous asthenia.

7,13

There was also

a low number of elastic bres and a disorganized elasticbre network as reported by Paciello and others.

11

Micro-haemorrhage in the dermis is a new nding, but has been

Figure 1. Skin biopsy specimens from the dorsum of a normal and an affected dog (haematoxylin and eosin). (a) Dermal collagen with normalappearance (bar = 50 m). (b) Dermal collagen is attenuated and disorganized (bar = 10 m). (c) Local haemorrhage (asterisks), increased vascu-larization (arrows) and broblast accumulation (arrowheads) (bar = 20 m).

Figure 2. Skin biopsy specimens from the dorsum of a normal and an affected dog (Massons trichrome). (a) Normal pattern of collagen bres(bar = 10 m). (b) Collagen bres exhibiting irregular dermal pattern, decrease in number and fragmentation (bar = 10 m).

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New ndings in canine cutaneous asthenia

reported in horses.

14

It may be secondary to trauma ondermal vessels, resulting from the excessive laxity of the

skin.Electron microscopy analysis showed a collagen abnor-mality known as spiralled collagen. Spiralled collagen is

thought to be the result of defective aggregation of collagenlaments into brils.

15

Such lesions have been described

in cattle and sheep with dermatosparaxis, in dogs, catsand mink with cutaneous asthenia, and in humans withEDS I and EDS IV.

1618

Figure 3. Electron microscopy of skin biopsy specimens from dorsum of a normal and an affected dog. (a) Normal dog skin note the parallel arrangementand transverse striations of the brils inside the collagen bres (bar = 0.5 m). (b) Affected dog skin the collagen brils are in disarray and spiralled, with ahighly irregular view in the transversal view (bar = 1m). (c) Affected dog skin showing thickening of the broblast nuclear lamina (arrow) (bar = 0.5 m).

Figure 4. Histological section of skin from the dorsum of a normal and an affected dog (Weigerts resorcinfuchsin). (a) Normal dog skin elastic bresare dispersed throughout the dermis (bar = 50 m). (b) Affected dog skin elastic bres are disorganized and decreased in number (bar = 50 m).

Figure 5. Electron microscopy of skin from the dorsum of affected dog. (a) Elastic bre with some deposition of elastineulanin bres (arrows)(bar = 0.5 m). (b) and (c) Fibres consisting of only microbrils oxytalan bres (bar = 0.5 m).

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The few elastic bres in the dermis of the affected dog wereconsisted of elaunin and oxytalan microbrils. These areimmature bres in which the brillar component is increasedbut elastin is reduced. It is possible that collagen bre failuretriggers a compensatory mechanism with broblasts syn-thesis of less elastic elastic bres to function as anchors.Quantitative changes in elastic tissues are considered indic-ative of the reduction of the collagen component and havebeen reported in cases of cutaneous asthenia and EDS IV.

10,19

In our study, broblasts exhibited conspicuous thickeningof the nuclear lamina. To the best of our knowledge, thisalteration has never been described in cutaneous astheniaand is perhaps the most important single nding from thisstudy. The nuclear brous lamina is composed of lamentsforming a bi-dimensional meshwork that underlies theinner nuclear membrane. These lamins provide structuralsupport for the nuclear membrane, allow chromatin attach-ment, and play a role in gene expression. Interactionsbetween lamins, lamin-associated proteins and chromatinseem to be important for certain nuclear functions.

20

Morphological alterations in the nuclear lamina havebeen described in 12 distinct human diseases. Thesediseases, collectively termed laminopathies, affect muscle,adipose, bone, nerve and skin cells, and range frommuscular dystrophies to accelerated ageing. The commonfeature of all these diseases is that they are related topremature ageing syndromes and that they display nuclearstructural abnormalities in broblasts harbouring mutationsin lamin or in other proteins associated with nuclear lamina.

21

HutchinsonGilford progeria syndrome is a rare sporadichuman disorder caused by mutations inLMNA

, the genethat encodes lamins A and C. Patients develop skin alterationssuch as hair greying, hair loss and skin thickening in their rst

few years of life. Dermal broblasts from these subjectsshow signicant changes in nuclear shape, thickening ofthe nuclear lamina, hypersensitivity to heat shock and delayedresponse to heat stress.

22,23

Another disease recently linkedto the lamina is focal dermal hypoplasia, a rare genoder-matosis characterized by a widespread dysplasia ofmesodermal and ectodermal tissues. This disorder primarilyaffects females, and may have an X-linked dominant mode ofinheritance. In affected skin areas, there are loosely arrangedcollagen bundles composed of few brils scattered in theextracellular matrix. Scarce elastic bres of normal morphol-ogy are also observed. Fibroblasts exhibit a remarkable andirregular thickening of the nuclear brous lamina.

24

A hypothesis to explain the differences among thelaminopathies is that the disruption of the nuclear laminaaffects chromatin organization and transcriptional regulationof gene expression in specic ways.

25

Broers et al. suggestedthat a common link might be a failure of stem cells toregenerate mesenchymal tissue. Thus, in canine cutaneousasthenia, this failure would induce the observed connectivetissue dysplasia.

20

Further studies are necessary toestablish a relationship between ultrastructural ndingsand the molecular abnormalities that cause this disease.

Acknowledgements

This study was supported by FAPESP (Process number:07/51204-4). The expert assistance of Dr Aline Trevisanwas greatly appreciated.

References

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11. Sequeira JL, Rocha NS, Bandarra EP, Figueiredo LM, Eugenio FR.Collagen dysplasia (cutaneous asthenia) in a cat. VeterinaryPathology 1999; 36: 6036.

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15. Ghadially FN. Ultrastructural Pathology of the Cell and Matrix, 4thedn. Boston, MA: Butterworths-Heinemann, 1997: 134850.

16. OHara PJ, Read WK, Romane WM, Brideges CH. A collagenoustissue dysplasia of calves. Laboratory Investigation 1970; 23:30714.

17. Patterson DF, Minor RR. Hereditary fragility and hyperextensibilityof the skin of cats. Laboratory Investigation 1977; 37: 1709.

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Rsum

Lasthnie cutane est une maladie gntique du tissu conjonctif, rencontre surtout chez lechien et le chat, ressemblant au syndrome dEhlers-Danlos de lhomme. La dysplasie collagnique rsulteen une hyperextensibilit cutane, une fragilit cutane et vasculaire et des troubles de la cicatrisation. Lebut de cette tude tait de dcrire les caractristiques histopathologiques chez un chien prsentant unecollagnopathie compatible avec une asthnie cutane. Une chienne crois ge de 8 mois a t prsentepour des lacrations et de nombreuses cicatrices atrophiques et irrgulires. La peau tait hyperextensibleet facilement dchire par le moindre traumatisme. Sur un plan ultrastructural, le derme tait constitu demicribrilles delaunine et doxytalan. Il sagit de bres immatures dans lesquelles la composantemicrobrillaire est augmente mais llastine est rduite. Les bres de collagne taient profondmentdsorganises. Les brilles avaient un aspect irrgulier et corrod en section transversale, et taient spiraleset fragmentes en section longitudinale. Les broblastes dermiques prsentaient un paississement de lalamina nuclaire. Les lamines nuclaires forment un rseau breux de laments de taille intermdiaire sousjacents la membrane nuclaire interne. Des mutations des lamines ou des protines associes aux laminesprovoquent une myriade de maladies gntiques appeles des laminopathies. La dsorganisation de lalamina nuclaire semble affecter lorganisation de la chromatine et la rgulation transcriptionnelle delexpression gnique. Un lien commun entre les laminopathies pourrait tre une incapacit des cellulessouches rgnrer le tissu msenchymateux. Ceci pourrait contribuer la dysplasie du tissu conjonctifoberve dans lasthnie cutane.

Resumen

La astenia cutnea es una enfermedad hereditaria del tejido conjuntivo, primariamente observadaen perros y gatos, y que semeja el sndrome de Ehlers-Danlos en humanos. Una displasia del colgenoresulta en hiperextensibilidad de la piel y en fragilidad de los vasos y una pobre cicatrizacin de heridas. Elpropsito de este estudio fue describir los hallazgos histopatolgicos en un perro con una colagenopataconsistente con astenia cutnea. Una perra mestiza de 8 meses se present con laceraciones y numerosascicatrices atrcas e irregulares. La piel era hiperextensible y se rompa fcilmente con mnimos traumas.A nivel ultraestructural la dermis estaba formada de microbrillas de elaunina y oxitaln. Estas sonbras inmaduras en las cuales el componente brilar est aumentado pero la elastina est reducida.Las bras de colgeno estaban intensamente desorganizadas. Las brillas tenan una estructura irregular yapariencia corroda cuando se observaban en secciones verticales, y eran espirales y fragmentadas envista longitudinal. Los broblastos de la dermis presentaban un engrosamiento conspicuo de la lminanuclear. Las lamininas nucleares forman una red estructural nuclear brosa de lamentos intermediosdebajo de la membrana nuclear interna. Mutaciones en las lamininas o en las protenas asociadas alamininas producen numerosas enfermedades genticas con denominacin colectiva de laminopatas.La disrupcin de la lmina nuclear parace afectar a la organizacin de la cromatina y la regulacin de latranscripcin en la expresin gnica. Un eslabn comn a todas la laminopatas puede ser un fallo de lasclulas madre para regenerar tejido mesenquimal. Esto podra contribuir a la displasia del tejido conjuntivoen la astenia cutnea.

Zusammenfassung

Die kutane Asthenie ist eine erbliche Krankheit des Bindegewebes, vor allembei Hunden und Katzen, welche dem Ehlers-Danlos Syndrom des Menschen hnlich ist. Die Dysplasiedes Kollagens resultiert in einer berdehnbarkeit der Haut, Haut- und Blutgeffragilitt, und schlechterWundheilung. Der Zweck dieser Studie war es, das histologischen Bild bei einem Hund mit einer Kollagen-opathie, die mit der kutanen Asthenie bereinstimmte, zu beschreiben. Eine 8 Monate alte Mischlingshndinwurde mit Risswunden und zahlreichen atrophischen und unregelmigen Narben vorgestellt. Die Haut warberdehnbar und riss beim geringsten Trauma. Ultrastrukturell bestand die Dermis aus Elaunin undOxytalan Mikrofasern. Es handelt sich dabei um unreife Fasern, in denen die brillre Komponente erhhtist, whrend das Elastin vermindert ist. Die Kollagenfasern waren hochgradig desorganisiert. Die Fibrillenhatten eine unregelmige Form und erschienen angefressen, wenn sie im Querschnitt betrachtet wurden,im Lngsschnitt waren sie gewunden und fragmentiert. Die dermalen Fibroblasten zeigten eine aufflligeVerdickung der nukleren Lamina. Die nukleren Lamine bilden ein brses nukleoskelettales Netzwerk vonFilamenten mittlerer Gre, die der inneren Kernmembran zugrunde liegen. Mutationen der Lamine oderder Lamin-assoziierten Proteine verursachen eine Unzahl von genetischen Erkrankungen, die zusammenals Laminopathien bezeichnet werden. Eine Strung der nukleren Lamina scheint die Ordnung desChromatins und die transkriptionale Regulierung der Genexpression zu beeintrchtigen. Eine Verbindungzwischen allen Laminopathien knnte das Versagen der Stammzellen sein mesenchymales Gewebe zuregenerieren. Das knnte zur Bindegewebsdysplasie, die bei der kutanen Asthenie gesehen wird, beitragen.

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