LMNA Mutations Induce a Non-Inflammatory Fibrosis and a Brown Fat-Like Dystrophy of Enlarged Cervical Adipose Tissue

• DOI: 10.1016/j.ajpath.2011.07.049
• Publication Date: 2011-09-22T15:13:33Z
• Authors: Véronique Béréziat; Pascale Cervera; Caroline Le Dour; Marie-Christine V...; Sylvie Dumont; Marie-Christine V...; Jacqueline Capeau; Corinne Vigouroux
• Keywords: LMNA; Lipodystrophy; Lipid droplet; Lipoatrophy

Some LMNA mutations responsible for insulin-resistant lipodystrophic syndromes are associated with peripheral subcutaneous lipoatrophy and faciocervical fat accumulation. Their pathophysiologic characteristics unknown. We compared histologic, immunohistologic, ultrastructural, protein expression features of enlarged cervical adipose tissue (scAT) obtained during plastic surgery from four patients p.R482W, p.R439C, or p.H506D versus eight control subjects, buffalo humps five HIV infection treated not protease inhibitors, dorsocervical lipomas two mitochondrial DNA mutations. LMNA-mutated scAT HIV-related were dystrophic, an increased percentage small adipocytes, fibrosis without inflammatory features, decreased number blood vessels, as samples. Samples inhibitor–based therapy demonstrated accumulation prelamin A, altered adipogenic proteins brown fat-like mitochondria overexpression uncoupling 1 (UCP1). These absent in samples subjects inhibitors. Mitochondrial DNA–mutated distinct abnormalities but neither UCP1 nor A In conclusion, Enlarged lipodystrophy fibrosis, vessel numbers. However, only who had received inhibitor accumulated exhibited similar remodeling toward a brown-like phenotype alterations. A-type lamins, encoded by the gene, ubiquitous nuclear intermediate filament involved structural functional integrity nucleus. Mutations cause inherited laminopathies including progeroid phenotypes lipodystrophies, metabolic alterations early cardiovascular complications (reviewed Mattout et al1Mattout A. Dechat T. Adam S.A. Goldman R.D. Gruenbaum Y. Nuclear diseases aging.Curr Opin Cell Biol. 2006; 18: 335-341Crossref PubMed Scopus (141) Google Scholar). Among them, Dunnigan-type familial partial (FPLD2; OMIM 151660), due primarily to p.R482 heterozygous substitutions, is characterized gradual atrophy extremities, trunk, gluteal areas, occurring after puberty, contrast excessive deposition face, chin, neck.2Cao H. Hegele R.A. lamin A/C R482Q mutation Canadian kindreds lipodystrophy.Hum Mol Genet. 2000; 9: 109-112Crossref (575) Scholar, 3Shackleton S. Lloyd D.J. Jackson S.N. Evans R. Niermeijer M.F. Singh B.M. Schmidt Brabant G. Kumar Durrington P.N. Gregory O'Rahilly Trembath R.C. LMNA, encoding A/C, mutated lipodystrophy.Nat 24: 153-156Crossref (593) Scholar The primary insulin resistance, impaired glucose tolerance diabetes, dyslipidemia marked hypertriglyceridemia.4Vigouroux C. Caron-Debarle M. Le Dour Magré J. Capeau Molecular mechanisms human lipodystrophies: adipocyte lipid droplet oxidative stress lipotoxicity.Int J Biochem 2011; 43: 862-876Crossref (106) 5Garg Agarwal A.K. Lipodystrophies: disorders biology.Biochim Biophys Acta. 2009; 1791: 507-513Crossref (143) Metabolic laminopathies, non–codon 482 mutations, severe atypical clinical lipoatrophy.6Decaudain Vantyghem M.C. Guerci B. Hecart A.C. Auclair Reznik Narbonne Ducluzeau P.H. Donadille Lebbé Béréziat V. Lascols O. Vigouroux New laminopathies: syndrome.J Clin Endocrinol Metab. 2007; 92: 4835-4844Crossref (118) Although fully understood, posttranslational maturation important pathogenic events.7Navarro C.L. Cau P. Lévy N. bases syndromes.Hum 15: R151-R161Crossref (147) 8Worman H.J. Fong L.G. Muchir Young S.G. Laminopathies long strange trip basic cell biology therapy.J Invest. 119: 1825-1836Crossref (213) 9Rodriguez Eriksson Evidence involvement lamins Aging Sci. 2010; 3: 81-89Crossref (15) Indeed, before being assembled lamina mature undergoes several steps addition farnesyl group followed proteolytic cleavage metalloprotease Zmpste24. others have that FPLD2 abnormal possibly misrecognition Zmpste24.10Capanni Mattioli E. Columbaro Lucarelli Parnaik V.K. Novelli Wehnert Cenni Maraldi N.M. Squarzoni Lattanzi Altered pre-lamin processing common mechanism leading 2005; 14: 1489-1502Crossref (181) 11Caron Laville Bodemer Human lipodystrophies linked both accumulation, premature cellular senescence.Cell Death Differ. 1759-1767Crossref (156) That can lead most depots, lipohypertrophy area, remains poorly understood could be differences depot physiologic features. It has been suggested may elicit different effects body depending on level local activation factor peroxisome proliferator–activated receptor-γ (PPARγ).10Capanni Partial (buffalo hump) also observed HIV-infected receiving antiretroviral therapy, thymidine analogues nucleoside reverse transcriptase inhibitors al12Caron-Debarle Lagathu Boccara F. HIV-associated lipodystrophy: injury aging.Trends Med. 16: 218-229Abstract Full Text PDF particular, ritonavir, widely used, induce accumulation,11Caron via direct inhibition zinc metallopeptidase Zmpste24.13Hudon S.E. Coffinier Michaelis Hrycyna C.A. HIV-protease block enzymatic activity purified Ste24p.Biochem Res Commun. 2008; 374: 365-368Crossref (42) Accordingly, presence lipoatrophic abdominal therapeutic regimen.11Caron previously reported cells and/or infection11Caron 14Villarroya Giralt Villarroya DNA: up-and-coming actor white pathophysiology.Obesity. 17: 1814-1820Crossref (30) 15De Pauw Tejerina Raes Keijer Arnould (dys)function (de)differentiation systemic alterations.Am Pathol. 175: 927-939Abstract (192) 16Hammond McKinnon Nolan D. immunodeficiency virus treatment-induced pathology lipoatrophy: prevalence consequences.Clin Infect Dis. 51: 591-599Crossref (55) Moreover, (mtDNA)–encoded tRNALys develop non-encapsulated masses,17Vila M.R. Gamez Solano Playan Schwartz Santorelli F.M. Cervera Casali Montoya Uncoupling protein-1 mRNA bearing mutations.Biochem 278: 800-802Crossref (32) 18Auré K. Sternberg Maisonobe Herson Jardel Blondy Lombès Eymard Laforêt Myopathy-lipomatosis A8344G mutation.Rev Neurol (Paris). 2003; 159: 1163-1168PubMed 19Guallar J.P. Vila Lopez-Gallardo Domingo J.C. Pineda Capablo J.L. Andreu A.L. master regulatory genes adipogenesis tRNA(Lys) point DNA.Mol Genet 89: 283-285Crossref (18) which suggests dysfunction role LMNA- HIV-linked lipodystrophies. Thus far, histologic reported, exception ultrastructural analysis revealed some adipocytes.20Araujo-Vilar Gonzalez-Mendez Costa-Freitas A.T. Prieto Victoria Martinez-Sanchez Ramazanova Fraga Beiras Forteza Dominguez-Gerpe L. Calvo Lado-Abeal Site-dependent type 2 lipodystrophy.J Med 46: 40-48Crossref (40) present work, we studied at levels. patients, mtDNA subjects. Cervical collected performed treat benign thyroid parotid (H-100 sc-7196) diabetes. Four women either p. R482W, typical phenotype,21Vigouroux Bourut Shackleton Padova Valensi Grimaldi Piquemal Touraine Lamin gene: sex-determined absence coding congenital acquired generalized lipoatrophy.Diabetes. 49: 1958-1962Crossref (153) 22Vantyghem Pigny Maurage Rouaix-Emery Stojkovic Cuisset J.M. Millaire Vermersch Wemeau Patients Dunnigan R482W show muscular cardiac abnormalities.J 2004; 5337-5346Crossref (105) (and unpublished data) p.R439C p.H506D, laminopathies.6Decaudain limb hypertrophy face neck developed progressively puberty. Lipodystrophy was resistance hypertriglyceridemia. humps) men currently (n = 2) 3) therapy. mixed fat. addition, unrelated referred because myopathy multiple lipomatosis m.8344A>G mutation18Auré data). Both underwent surgical removal large lipoma, clinically hump.18Auré Characteristics given Table 1. Informed consent all according our ethics committee.Table 1Characteristics ControlsSexAge (years)BMILocalization fatLipodystrophyDiabetesHOMATriglycerides (mmol/L)Nonalcoholic steatohepatitisOtherControlsF4824.5Anterior neckNoNoNANANANA M6923.9Lateral M4027.5Anterior F5422.1Anterior neckNoNo0.70.75NANA M8224.8Lateral neckNoNo1.41.5NANA F8723Anterior neckNoNo1.31.2NANA F6527.3Anterior neckNoNo3.91.3NANA M6223Anterior neckNoNo1.52.7NANAPatients F⁎p.R482W.2128.7Anterior neckLipoatrophy lower limbs, accumulationYes18.13.7NANo F⁎p.R482W.2823Anterior limbs accumulationYes13.53YesNo F†p.R439C3328.3Anterior accumulationImpaired tolerance4.31.8YesPelvic muscle weakness F‡p.H506D5622.7Anterior fasting glucose3.52.6NoNoHIV-infected patientsCurrent ARV M5923.1DorsocervicalLipoatrophy neckNoNANANATDF, 3TC, ABC, DRV, RTV M4323.7DorsocervicalLipoatrophy neckNoNANANAd4T, IDV, M5524.8DorsocervicalBuffalo neckNoNANANAZDV, NVP M5526.4DorsocervicalBuffalo neckNo1.82.2-TDF, ABC M4441.3DorsocervicalBuffalo neckYesInsulin therapy2.5YesNVP, MVC, RALPatients M5233.9DorsocervicalLipomas neck, shouldersNoNA1.7NAMyopathy, sensory polyneuropathy M5426DorsocervicalLipomas scalp, calves, thoraxNoNANANoMyopathyF, female; M, male; ARV, drug; HOMA-IR, homeostasis model assessment resistance; NA, available. Nucleoside nucleotide inhibitors: TDF, tenofovir; lamivudine; abacavir; d4T, stavudine; ZDV, zidovudine. Non-nucleoside inhibitor: NVP, nevirapine. Protease darunavir; RTV, ritonavi; indinavir. CCR5 maraviroc. Integrase RAL, raltegravir. p.R482W.† p.R439C‡ Open table new tab F, Light microscopy immunohistochemical studies described.23Jan Maachi Baudrimont Kim Vidal Girard P.M. Levan Rozenbaum W. Bastard differentiation adipocytokine inter-related morphological changes HIV-1-infected patients.Antivir Ther. 555-564PubMed brief, light using 10% zinc-formol–fixed paraffin-embedded 5-mm sections stained hemalum-phloxine morphologic Sirius Red detect collagen fibers. mean index lipogranulomas determined semi-automatic image system (Mercator; Explora Nova, La Rochelle, France) three randomly chosen regions. ratio total surfaces defined fibrosis. For studies, probed antibodies directed against proinflammatory macrophages marker CD6823Jan 24Nolan Hammond Martin Taylor Herrmann Metcalf Latham Mallal depletion adipocytes therapy.AIDS. 1329-1338Crossref (235) 25Kim M.J. Leclercq Lanoy Antuna-Puente Dorofeev Slama Valantin M.A. Costagliola 6-month interruption improves function patients: ANRS EP29 Lipostop Study.Antivir 12: 1273-1283PubMed (1:300; Dako Corp., Carpenteria, CA), cytokines IL-6 (IL6, MAB206 1:50) tumor necrosis factor-α (h-TNFα, MAB610 (both R&D Systems Europe, Ltd., Abingdon, Oxfordshire, England), respiratory chain cytochrome c oxidase subunits (COX2) 4 (COX4) (A-6404, 1:100 A-21348, 1:100; Probes, Inc., Eugene, OR), antigen (MU213-UC, 1:50; BioGenex Laboratories, San Ramon, vascular CD34 (endothelial cells) (clone QBEnd-10) α-smooth actin (α-SMA, staining media layer arteries, clone 1A4) SA, Trappes, France). Ultrastructural fixed 2.5% glutaraldehyde 0.1 mmol/L cacodylate buffer (pH 7.4) 4°C. Fragments then post-fixed 1% osmium tetroxide, dehydrated graded alcohol series, embedded epoxy resin. Semi-fine (0.5 μm) toluidine blue. Ultrastructure (60 nm) contrast-enhanced uranyl acetate citrate, examined JEOL 1010 electron microscope (JEOL, Tokyo, Japan) MegaView III camera (Olympus Soft Imaging GmbH, Münster, Germany). Total RNA extracted stored liquid nitrogen RNeasy Lipid Tissue Minikit (Qiagen Courtaboeuf, measured real-time RT-PCR LightCycler (Roche Diagnostics France Meylan, France), described.25Kim following primers used: TATA-binding (used internal standard expression): forward, 5′-GCTCACCCACCAACAATTTAG-3′, reverse, 5′-GAGCCATTACGTCGTCTTCC-3′; CD68: 5′-TCAGCTTTGGATTCATGCAG-3′, 5′-AGGTGGACAGCTGGTGAAAG-3′. Frozen (300 mg) solubilized 500 μL 2.5X Laemmli containing 150 dithiotreitol. Lysates subjected SDS-PAGE, blotted onto nitrocellulose membranes, transcription factors PPARγ, sterol element-binding (SREBP-1) (K-10 sc-367) (sc-6214) (all Santa Cruz Biotechnology, Cruz, (MAB-3211; Chemicon International, Temecula, COX2 COX4 (A-6404 Inc.), voltage-dependent anion channel (VDAC/porin, Ab-5 PC548; Merck KGaA, Darmstadt, Germany), (ab10983; Abcam, Cambridge, England). detected chemiluminescence detection kit (Amersham Pharmacia Biotech Les Ulis, β-Actin (A5441; Sigma-Aldrich St. Louis, MO) used level. Quantifications, normalized β-actin expression, ChemiGenius2 analyzer software (Ozyme, Quentin en Yvelines, expressed fold control. All results triplicate experiments, ± SEM. controls, composed lobules univacuolar delineated sharp thin capsules evidenced staining. disposition size overall homogeneous (Figure 1). contrast, infection, not, heterogeneous structure clusters normal-sized fibrotic bundles thickening areas did represent scar undergone previous surgery. agreement these observations, patient than (ie, 1734, 1968, 1692 μm2 respectively, 2988 subjects) 2A). higher 44.5, 25.6, 17.5 2.9 2B). At level, regular cytoplasm intercellular areas. irregular outlines observed, thickened rims interstitial edema compact thick fibrils invading area 3). lipodystrophy, rare observed. cases, few CD68, 4A). CD68 labeling TNF-α (data shown). Conversely, mtDNA-mutated lipomas, disrupted variably fragmented cytoplasmic rims, droplets lying free connective tissue, numerous samples, many lipogranulomas, ie, CD68-positive organized into crown-like structures surrounding involutionary 4, B), data mutations,20Araujo-Vilar nucleus architecture Considered together, demonstrate dystrophic infection. As shown Figure 5, surface occupied vessels assessed immunohistologic endothelial CD34. α-SMA, shown, 5B). when evaluated arteries excluding SREBP-1 PPARγ same pattern inhibitor–treated infection: underexpressed 6). current treatment, significantly differ