LMNA Mutations Induce a Non-Inflammatory Fibrosis and a Brown Fat-Like Dystrophy of Enlarged Cervical Adipose Tissue
2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.07.049
ISSN1525-2191
AutoresVéronique Béréziat, Pascale Cervera, Caroline Le Dour, Marie‐Christine Verpont, Sylvie Dumont, Marie‐Christine Vantyghem, Jacqueline Capeau, Corinne Vigouroux,
Tópico(s)Muscle Physiology and Disorders
ResumoSome LMNA mutations responsible for insulin-resistant lipodystrophic syndromes are associated with peripheral subcutaneous lipoatrophy and faciocervical fat accumulation. Their pathophysiologic characteristics are unknown. We compared histologic, immunohistologic, ultrastructural, and protein expression features of enlarged cervical subcutaneous adipose tissue (scAT) obtained during plastic surgery from four patients with LMNA p.R482W, p.R439C, or p.H506D mutations versus cervical fat from eight control subjects, buffalo humps from five patients with HIV infection treated or not with protease inhibitors, and dorsocervical lipomas from two patients with mitochondrial DNA mutations. LMNA-mutated cervical scAT and HIV-related buffalo humps were dystrophic, with an increased percentage of small adipocytes, increased fibrosis without inflammatory features, and decreased number of blood vessels, as compared with control samples. Samples from patients with LMNA mutations or protease inhibitor–based therapy demonstrated accumulation of prelamin A, altered expression of adipogenic proteins and brown fat-like features, with an increased number of mitochondria and overexpression of uncoupling protein 1 (UCP1). These features were absent in samples from control subjects and from patients with HIV not treated with protease inhibitors. Mitochondrial DNA–mutated cervical lipomas demonstrated inflammatory fibrosis with distinct mitochondrial abnormalities but neither UCP1 expression nor prelamin A accumulation. In conclusion, Enlarged cervical scAT from patients with lipodystrophy demonstrated small adipocytes, fibrosis, and decreased vessel numbers. However, only cervical fat from patients with LMNA mutations or who had received protease inhibitor therapy accumulated prelamin A and exhibited similar remodeling toward a brown-like phenotype with UCP1 overexpression and mitochondrial alterations. Some LMNA mutations responsible for insulin-resistant lipodystrophic syndromes are associated with peripheral subcutaneous lipoatrophy and faciocervical fat accumulation. Their pathophysiologic characteristics are unknown. We compared histologic, immunohistologic, ultrastructural, and protein expression features of enlarged cervical subcutaneous adipose tissue (scAT) obtained during plastic surgery from four patients with LMNA p.R482W, p.R439C, or p.H506D mutations versus cervical fat from eight control subjects, buffalo humps from five patients with HIV infection treated or not with protease inhibitors, and dorsocervical lipomas from two patients with mitochondrial DNA mutations. LMNA-mutated cervical scAT and HIV-related buffalo humps were dystrophic, with an increased percentage of small adipocytes, increased fibrosis without inflammatory features, and decreased number of blood vessels, as compared with control samples. Samples from patients with LMNA mutations or protease inhibitor–based therapy demonstrated accumulation of prelamin A, altered expression of adipogenic proteins and brown fat-like features, with an increased number of mitochondria and overexpression of uncoupling protein 1 (UCP1). These features were absent in samples from control subjects and from patients with HIV not treated with protease inhibitors. Mitochondrial DNA–mutated cervical lipomas demonstrated inflammatory fibrosis with distinct mitochondrial abnormalities but neither UCP1 expression nor prelamin A accumulation. In conclusion, Enlarged cervical scAT from patients with lipodystrophy demonstrated small adipocytes, fibrosis, and decreased vessel numbers. However, only cervical fat from patients with LMNA mutations or who had received protease inhibitor therapy accumulated prelamin A and exhibited similar remodeling toward a brown-like phenotype with UCP1 overexpression and mitochondrial alterations. A-type lamins, encoded by the LMNA gene, are ubiquitous nuclear intermediate filament proteins involved in the structural and functional integrity of the nucleus. Mutations in LMNA cause inherited laminopathies including progeroid phenotypes and lipodystrophies, with metabolic alterations and early cardiovascular complications (reviewed in Mattout et al1Mattout A. Dechat T. Adam S.A. Goldman R.D. Gruenbaum Y. Nuclear lamins, diseases and aging.Curr Opin Cell Biol. 2006; 18: 335-341Crossref PubMed Scopus (141) Google Scholar). Among them, the Dunnigan-type familial partial lipodystrophy (FPLD2; OMIM 151660), due primarily to LMNA p.R482 heterozygous substitutions, is characterized by gradual atrophy of subcutaneous adipose tissue (scAT) in the extremities, trunk, and gluteal areas, occurring after puberty, in contrast to excessive adipose tissue deposition in the face, chin, and neck.2Cao H. Hegele R.A. Nuclear lamin A/C R482Q mutation in Canadian kindreds with Dunnigan-type familial partial lipodystrophy.Hum Mol Genet. 2000; 9: 109-112Crossref PubMed Scopus (575) Google Scholar, 3Shackleton S. Lloyd D.J. Jackson S.N. Evans R. Niermeijer M.F. Singh B.M. Schmidt H. Brabant G. Kumar S. Durrington P.N. Gregory S. O'Rahilly S. Trembath R.C. LMNA, encoding lamin A/C, is mutated in partial lipodystrophy.Nat Genet. 2000; 24: 153-156Crossref PubMed Scopus (593) Google Scholar The primary metabolic alterations are insulin resistance, impaired glucose tolerance or diabetes, and dyslipidemia with marked hypertriglyceridemia.4Vigouroux C. Caron-Debarle M. Le Dour C. Magré J. Capeau J. Molecular mechanisms of human lipodystrophies: from adipocyte lipid droplet to oxidative stress and lipotoxicity.Int J Biochem Cell Biol. 2011; 43: 862-876Crossref PubMed Scopus (106) Google Scholar, 5Garg A. Agarwal A.K. Lipodystrophies: disorders of adipose tissue biology.Biochim Biophys Acta. 2009; 1791: 507-513Crossref PubMed Scopus (143) Google Scholar Metabolic laminopathies, due to non–codon 482 LMNA mutations, are characterized by severe metabolic alterations but atypical clinical lipoatrophy.6Decaudain A. Vantyghem M.C. Guerci B. Hecart A.C. Auclair M. Reznik Y. Narbonne H. Ducluzeau P.H. Donadille B. Lebbé C. Béréziat V. Capeau J. Lascols O. Vigouroux C. New metabolic phenotypes in laminopathies: LMNA mutations in patients with severe metabolic syndrome.J Clin Endocrinol Metab. 2007; 92: 4835-4844Crossref PubMed Scopus (118) Google Scholar Although the pathophysiologic mechanisms involved in laminopathies are not fully understood, alterations in the posttranslational maturation of prelamin A are important pathogenic events.7Navarro C.L. Cau P. Lévy N. Molecular bases of progeroid syndromes.Hum Mol Genet. 2006; 15: R151-R161Crossref PubMed Scopus (147) Google Scholar, 8Worman H.J. Fong L.G. Muchir A. Young S.G. Laminopathies and the long strange trip from basic cell biology to therapy.J Clin Invest. 2009; 119: 1825-1836Crossref PubMed Scopus (213) Google Scholar, 9Rodriguez S. Eriksson M. Evidence for the involvement of lamins in aging.Curr Aging Sci. 2010; 3: 81-89Crossref PubMed Scopus (15) Google Scholar Indeed, before being assembled in the nuclear lamina as mature lamin A, prelamin A undergoes several maturation steps including addition of a farnesyl group followed by a proteolytic cleavage by the metalloprotease Zmpste24. We and others have demonstrated that FPLD2 and metabolic laminopathies are associated with an abnormal accumulation of prelamin A, possibly due to misrecognition of the mutated protein by Zmpste24.10Capanni C. Mattioli E. Columbaro M. Lucarelli E. Parnaik V.K. Novelli G. Wehnert M. Cenni V. Maraldi N.M. Squarzoni S. Lattanzi G. Altered pre-lamin A processing is a common mechanism leading to lipodystrophy.Hum Mol Genet. 2005; 14: 1489-1502Crossref PubMed Scopus (181) Google Scholar, 11Caron M. Auclair M. Donadille B. Béréziat V. Guerci B. Laville M. Narbonne H. Bodemer C. Lascols O. Capeau J. Vigouroux C. Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence.Cell Death Differ. 2007; 14: 1759-1767Crossref PubMed Scopus (156) Google Scholar That LMNA mutations can lead to lipoatrophy in most scAT depots, but to lipohypertrophy in the faciocervical area, remains poorly understood but could be linked to differences in fat depot physiologic features. It has been suggested that prelamin A accumulation may elicit different effects in body fat areas, depending on the level of local activation of the adipogenic factor peroxisome proliferator–activated receptor-γ (PPARγ).10Capanni C. Mattioli E. Columbaro M. Lucarelli E. Parnaik V.K. Novelli G. Wehnert M. Cenni V. Maraldi N.M. Squarzoni S. Lattanzi G. Altered pre-lamin A processing is a common mechanism leading to lipodystrophy.Hum Mol Genet. 2005; 14: 1489-1502Crossref PubMed Scopus (181) Google Scholar Partial lipodystrophies with peripheral lipoatrophy but increased cervical fat (buffalo hump) are also observed in HIV-infected patients receiving antiretroviral therapy, primarily the thymidine analogues nucleoside reverse transcriptase inhibitors and HIV protease inhibitors (reviewed in Caron-Debarle et al12Caron-Debarle M. Lagathu C. Boccara F. Vigouroux C. Capeau J. HIV-associated lipodystrophy: from fat injury to premature aging.Trends Mol Med. 2010; 16: 218-229Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Some protease inhibitors, in particular, ritonavir, widely used, can induce cellular prelamin A accumulation,11Caron M. Auclair M. Donadille B. Béréziat V. Guerci B. Laville M. Narbonne H. Bodemer C. Lascols O. Capeau J. Vigouroux C. Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence.Cell Death Differ. 2007; 14: 1759-1767Crossref PubMed Scopus (156) Google Scholar via direct inhibition of the zinc metallopeptidase Zmpste24.13Hudon S.E. Coffinier C. Michaelis S. Fong L.G. Young S.G. Hrycyna C.A. HIV-protease inhibitors block the enzymatic activity of purified Ste24p.Biochem Biophys Res Commun. 2008; 374: 365-368Crossref PubMed Scopus (42) Google Scholar Accordingly, the presence of prelamin A has been observed in lipoatrophic abdominal scAT from HIV-infected patients receiving a protease inhibitor–based therapeutic regimen.11Caron M. Auclair M. Donadille B. Béréziat V. Guerci B. Laville M. Narbonne H. Bodemer C. Lascols O. Capeau J. Vigouroux C. Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence.Cell Death Differ. 2007; 14: 1759-1767Crossref PubMed Scopus (156) Google Scholar We and others have previously reported the presence of mitochondrial abnormalities in cells and/or lipoatrophic adipose tissue from patients with LMNA mutations or HIV infection11Caron M. Auclair M. Donadille B. Béréziat V. Guerci B. Laville M. Narbonne H. Bodemer C. Lascols O. Capeau J. Vigouroux C. Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence.Cell Death Differ. 2007; 14: 1759-1767Crossref PubMed Scopus (156) Google Scholar, 14Villarroya J. Giralt M. Villarroya F. Mitochondrial DNA: an up-and-coming actor in white adipose tissue pathophysiology.Obesity. 2009; 17: 1814-1820Crossref PubMed Scopus (30) Google Scholar, 15De Pauw A. Tejerina S. Raes M. Keijer J. Arnould T. Mitochondrial (dys)function in adipocyte (de)differentiation and systemic metabolic alterations.Am J Pathol. 2009; 175: 927-939Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 16Hammond E. McKinnon E. Nolan D. Human immunodeficiency virus treatment-induced adipose tissue pathology and lipoatrophy: prevalence and metabolic consequences.Clin Infect Dis. 2010; 51: 591-599Crossref PubMed Scopus (55) Google Scholar Moreover, patients with mutations in mitochondrial DNA (mtDNA)–encoded tRNALys can develop dorsocervical non-encapsulated fat masses,17Vila M.R. Gamez J. Solano A. Playan A. Schwartz S. Santorelli F.M. Cervera C. Casali C. Montoya J. Villarroya F. Uncoupling protein-1 mRNA expression in lipomas from patients bearing pathogenic mitochondrial DNA mutations.Biochem Biophys Res Commun. 2000; 278: 800-802Crossref PubMed Scopus (32) Google Scholar, 18Auré K. Sternberg D. Maisonobe T. Herson S. Jardel C. Blondy P. Lombès A. Eymard B. Laforêt P. Myopathy-lipomatosis associated with A8344G mitochondrial DNA mutation.Rev Neurol (Paris). 2003; 159: 1163-1168PubMed Google Scholar, 19Guallar J.P. Vila M.R. Lopez-Gallardo E. Solano A. Domingo J.C. Gamez J. Pineda M. Capablo J.L. Domingo P. Andreu A.L. Montoya J. Giralt M. Villarroya F. Altered expression of master regulatory genes of adipogenesis in lipomas from patients bearing tRNA(Lys) point mutations in mitochondrial DNA.Mol Genet Metab. 2006; 89: 283-285Crossref PubMed Scopus (18) Google Scholar which suggests that mitochondrial dysfunction could also have a role in LMNA- and HIV-linked lipodystrophies. Thus far, histologic features of LMNA-mutated scAT have not been reported, with the exception of an ultrastructural analysis that revealed nuclear alterations in some lipoatrophic adipocytes.20Araujo-Vilar D. Lattanzi G. Gonzalez-Mendez B. Costa-Freitas A.T. Prieto D. Columbaro M. Mattioli E. Victoria B. Martinez-Sanchez N. Ramazanova A. Fraga M. Beiras A. Forteza J. Dominguez-Gerpe L. Calvo C. Lado-Abeal J. Site-dependent differences in both prelamin A and adipogenic genes in subcutaneous adipose tissue of patients with type 2 familial partial lipodystrophy.J Med Genet. 2009; 46: 40-48Crossref PubMed Scopus (40) Google Scholar In the present work, we studied alterations of enlarged cervical adipose tissue from patients with LMNA mutations at the histologic, immunohistologic, ultrastructural, and protein expression levels. These fat samples were compared with buffalo humps from HIV-infected patients, treated or not with protease inhibitors, with cervical lipomas due to mtDNA mutations and with cervical fat from control subjects. Cervical scAT samples were collected during plastic surgery in patients and during surgery performed to treat benign thyroid or parotid diseases (H-100 sc-7196) in eight control patients without diabetes. Four women had heterozygous LMNA mutations, either p. R482W, leading to a typical FPLD2 phenotype,21Vigouroux C. Magré J. Vantyghem M.C. Bourut C. Lascols O. Shackleton S. Lloyd D.J. Guerci B. Padova G. Valensi P. Grimaldi A. Piquemal R. Touraine P. Trembath R.C. Capeau J. Lamin A/C gene: sex-determined expression of mutations in Dunnigan-type familial partial lipodystrophy and absence of coding mutations in congenital and acquired generalized lipoatrophy.Diabetes. 2000; 49: 1958-1962Crossref PubMed Scopus (153) Google Scholar, 22Vantyghem M.C. Pigny P. Maurage C.A. Rouaix-Emery N. Stojkovic T. Cuisset J.M. Millaire A. Lascols O. Vermersch P. Wemeau J.L. Capeau J. Vigouroux C. Patients with familial partial lipodystrophy of the Dunnigan type due to a LMNA R482W mutation show muscular and cardiac abnormalities.J Clin Endocrinol Metab. 2004; 89: 5337-5346Crossref PubMed Scopus (105) Google Scholar (and unpublished data) or p.R439C or p.H506D, leading to metabolic laminopathies.6Decaudain A. Vantyghem M.C. Guerci B. Hecart A.C. Auclair M. Reznik Y. Narbonne H. Ducluzeau P.H. Donadille B. Lebbé C. Béréziat V. Capeau J. Lascols O. Vigouroux C. New metabolic phenotypes in laminopathies: LMNA mutations in patients with severe metabolic syndrome.J Clin Endocrinol Metab. 2007; 92: 4835-4844Crossref PubMed Scopus (118) Google Scholar These four women demonstrated marked subcutaneous limb lipoatrophy with muscular hypertrophy and fat accumulation in the face and neck that had developed progressively after puberty. Lipodystrophy was associated with insulin resistance and hypertriglyceridemia. We also collected accumulated dorsocervical fat samples (buffalo humps) from five HIV-infected men currently receiving (n = 2) or not receiving (n = 3) protease inhibitor–based antiretroviral therapy. These patients developed mixed lipodystrophy with peripheral lipoatrophy but increased dorsocervical fat. In addition, two unrelated men were referred because of myopathy and multiple lipomatosis due to the mtDNA tRNALys m.8344A>G mutation18Auré K. Sternberg D. Maisonobe T. Herson S. Jardel C. Blondy P. Lombès A. Eymard B. Laforêt P. Myopathy-lipomatosis associated with A8344G mitochondrial DNA mutation.Rev Neurol (Paris). 2003; 159: 1163-1168PubMed Google Scholar (and unpublished data). Both men underwent surgical removal of a large dorsocervical lipoma, clinically similar to a buffalo hump.18Auré K. Sternberg D. Maisonobe T. Herson S. Jardel C. Blondy P. Lombès A. Eymard B. Laforêt P. Myopathy-lipomatosis associated with A8344G mitochondrial DNA mutation.Rev Neurol (Paris). 2003; 159: 1163-1168PubMed Google Scholar Characteristics of patients and control subjects are given in Table 1. Informed consent was obtained from all patients and control subjects, according to our local ethics committee.Table 1Characteristics of Patients and ControlsSexAge (years)BMILocalization of collected fatLipodystrophyDiabetesHOMATriglycerides (mmol/L)Nonalcoholic steatohepatitisOtherControlsF4824.5Anterior neckNoNoNANANANA M6923.9Lateral neckNoNoNANANANA M4027.5Anterior neckNoNoNANANANA F5422.1Anterior neckNoNo0.70.75NANA M8224.8Lateral neckNoNo1.41.5NANA F8723Anterior neckNoNo1.31.2NANA F6527.3Anterior neckNoNo3.91.3NANA M6223Anterior neckNoNo1.52.7NANAPatients with LMNA mutations F⁎p.R482W.2128.7Anterior neckLipoatrophy of lower limbs, faciocervical fat accumulationYes18.13.7NANo F⁎p.R482W.2128.7Anterior neckLipoatrophy of lower limbs, faciocervical fat accumulationYes18.13.7NANo F⁎p.R482W.2823Anterior neckLipoatrophy of lower limbs and trunk, faciocervical fat accumulationYes13.53YesNo F†p.R439C3328.3Anterior neckLipoatrophy of lower limbs, faciocervical fat accumulationImpaired glucose tolerance4.31.8YesPelvic muscle weakness F‡p.H506D5622.7Anterior neckLipoatrophy of lower limbs, faciocervical fat accumulationImpaired fasting glucose3.52.6NoNoHIV-infected patientsCurrent ARV M5923.1DorsocervicalLipoatrophy of limbs, buffalo neckNoNANANATDF, 3TC, ABC, DRV, RTV M4323.7DorsocervicalLipoatrophy of limbs, buffalo neckNoNANANAd4T, 3TC, IDV, RTV M5524.8DorsocervicalBuffalo neckNoNANANAZDV, 3TC, NVP M5526.4DorsocervicalBuffalo neckNo1.82.2-TDF, 3TC, ABC M4441.3DorsocervicalBuffalo neckYesInsulin therapy2.5YesNVP, MVC, RALPatients with tRNALys m.8344A>G mtDNA mutation M5233.9DorsocervicalLipomas in neck, chin, and shouldersNoNA1.7NAMyopathy, sensory polyneuropathy M5426DorsocervicalLipomas in neck, scalp, chin, calves, and thoraxNoNANANoMyopathyF, female; M, male; ARV, antiretroviral drug; HOMA-IR, homeostasis model assessment of insulin resistance; NA, not available. Nucleoside or nucleotide reverse transcriptase inhibitors: TDF, tenofovir; 3TC, lamivudine; ABC, abacavir; d4T, stavudine; ZDV, zidovudine. Non-nucleoside reverse transcriptase inhibitor: NVP, nevirapine. Protease inhibitors: DRV, darunavir; RTV, ritonavi; IDV, indinavir. CCR5 inhibitor: MVC, maraviroc. Integrase inhibitor: RAL, raltegravir. p.R482W.† p.R439C‡ p.H506D Open table in a new tab F, female; M, male; ARV, antiretroviral drug; HOMA-IR, homeostasis model assessment of insulin resistance; NA, not available. Nucleoside or nucleotide reverse transcriptase inhibitors: TDF, tenofovir; 3TC, lamivudine; ABC, abacavir; d4T, stavudine; ZDV, zidovudine. Non-nucleoside reverse transcriptase inhibitor: NVP, nevirapine. Protease inhibitors: DRV, darunavir; RTV, ritonavi; IDV, indinavir. CCR5 inhibitor: MVC, maraviroc. Integrase inhibitor: RAL, raltegravir. Light microscopy and immunohistochemical studies were performed as previously described.23Jan V. Cervera P. Maachi M. Baudrimont M. Kim M. Vidal H. Girard P.M. Levan P. Rozenbaum W. Lombès A. Capeau J. Bastard J.P. Altered fat differentiation and adipocytokine expression are inter-related and linked to morphological changes and insulin resistance in HIV-1-infected lipodystrophic patients.Antivir Ther. 2004; 9: 555-564PubMed Google Scholar In brief, light microscopy was performed using 10% zinc-formol–fixed paraffin-embedded 5-mm tissue sections stained with hemalum-phloxine for morphologic studies and with Sirius Red to detect collagen fibers. The adipocyte mean areas, index of fibrosis, and number of lipogranulomas were determined using a semi-automatic image analysis system (Mercator; Explora Nova, La Rochelle, France) in three randomly chosen regions. The ratio of fibrosis to total adipose tissue surfaces defined the index of fibrosis. For immunohistochemical studies, tissue sections were probed using antibodies directed against the proinflammatory macrophages marker CD6823Jan V. Cervera P. Maachi M. Baudrimont M. Kim M. Vidal H. Girard P.M. Levan P. Rozenbaum W. Lombès A. Capeau J. Bastard J.P. Altered fat differentiation and adipocytokine expression are inter-related and linked to morphological changes and insulin resistance in HIV-1-infected lipodystrophic patients.Antivir Ther. 2004; 9: 555-564PubMed Google Scholar, 24Nolan D. Hammond E. Martin A. Taylor L. Herrmann S. McKinnon E. Metcalf C. Latham B. Mallal S. Mitochondrial DNA depletion and morphologic changes in adipocytes associated with nucleoside reverse transcriptase inhibitor therapy.AIDS. 2003; 17: 1329-1338Crossref PubMed Scopus (235) Google Scholar, 25Kim M.J. Leclercq P. Lanoy E. Cervera P. Antuna-Puente B. Maachi M. Dorofeev E. Slama L. Valantin M.A. Costagliola D. Lombès A. Bastard J.P. Capeau J. A 6-month interruption of antiretroviral therapy improves adipose tissue function in HIV-infected patients: the ANRS EP29 Lipostop Study.Antivir Ther. 2007; 12: 1273-1283PubMed Google Scholar (1:300; Dako Corp., Carpenteria, CA), the inflammatory cytokines IL-6 (IL6, MAB206 1:50) and tumor necrosis factor-α (h-TNFα, MAB610 1:50) (both from R&D Systems Europe, Ltd., Abingdon, Oxfordshire, England), the mitochondrial respiratory chain proteins cytochrome c oxidase subunits 2 (COX2) and 4 (COX4) (A-6404, 1:100 and A-21348, 1:100; Molecular Probes, Inc., Eugene, OR), a mitochondrial antigen (MU213-UC, 1:50; BioGenex Laboratories, Inc., San Ramon, CA), and the vascular marker CD34 (endothelial cells) (clone QBEnd-10) and α-smooth muscle actin (α-SMA, staining the media layer of arteries, clone 1A4) (both from Dako SA, Trappes, France). Ultrastructural analysis was performed on fat samples fixed in 2.5% glutaraldehyde in 0.1 mmol/L cacodylate buffer (pH 7.4) at 4°C. Fragments were then post-fixed in 1% osmium tetroxide, dehydrated using graded alcohol series, and embedded in epoxy resin. Semi-fine sections (0.5 μm) were stained using toluidine blue. Ultrastructure sections (60 nm) were contrast-enhanced using uranyl acetate and lead citrate, and examined using a JEOL 1010 electron microscope (JEOL, Ltd., Tokyo, Japan) with a MegaView III camera (Olympus Soft Imaging Systems GmbH, Münster, Germany). Total RNA was extracted from samples stored in liquid nitrogen using the RNeasy Lipid Tissue Minikit (Qiagen SA, Courtaboeuf, France). mRNA expression was measured using real-time RT-PCR on the LightCycler system (Roche Diagnostics France SA, Meylan, France), as previously described.25Kim M.J. Leclercq P. Lanoy E. Cervera P. Antuna-Puente B. Maachi M. Dorofeev E. Slama L. Valantin M.A. Costagliola D. Lombès A. Bastard J.P. Capeau J. A 6-month interruption of antiretroviral therapy improves adipose tissue function in HIV-infected patients: the ANRS EP29 Lipostop Study.Antivir Ther. 2007; 12: 1273-1283PubMed Google Scholar The following primers were used: for TATA-binding protein (used as an internal standard for mRNA expression): forward, 5′-GCTCACCCACCAACAATTTAG-3′, and reverse, 5′-GAGCCATTACGTCGTCTTCC-3′; and for CD68: forward, 5′-TCAGCTTTGGATTCATGCAG-3′, and reverse, 5′-AGGTGGACAGCTGGTGAAAG-3′. Frozen fat tissue (300 mg) was solubilized in 500 μL 2.5X Laemmli buffer containing 150 mmol/L dithiotreitol. Lysates were subjected to SDS-PAGE, blotted onto nitrocellulose membranes, and probed using antibodies directed against the adipocyte transcription factors PPARγ, sterol regulatory element-binding protein 1 (SREBP-1) (K-10 sc-367) and prelamin A (sc-6214) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), lamin A/C (MAB-3211; Chemicon International, Inc., Temecula, CA), COX2 and COX4 (A-6404 1:100 and A-21348, 1:100; Molecular Probes, Inc.), mitochondrial voltage-dependent anion channel (VDAC/porin, Ab-5 PC548; Merck KGaA, Darmstadt, Germany), and UCP1 (ab10983; Abcam, Ltd., Cambridge, England). The antibodies were detected using a chemiluminescence detection kit (Amersham Pharmacia Biotech SA, Les Ulis, France). β-Actin (A5441; Sigma-Aldrich Corp., St. Louis, MO) was used as an index of the cellular protein level. Quantifications, normalized to β-actin expression, were performed by using the ChemiGenius2 image analyzer and software (Ozyme, St. Quentin en Yvelines, France), and were expressed in fold changes versus control. All results were from triplicate experiments, and are expressed as mean ± SEM. In the eight controls, fat samples were composed of several lobules with mature univacuolar adipocytes delineated by sharp thin capsules without fibrosis, as evidenced at Sirius Red staining. The disposition and size of control adipocytes were overall homogeneous (Figure 1). In contrast, adipose tissue from patients with LMNA mutations, HIV infection, receiving protease inhibitor therapy or not, or mtDNA mutations had a heterogeneous structure with clusters of small or normal-sized adipocytes (Figure 1). In addition, marked fibrosis was present in cervical scAT from all patients and demonstrated fibrotic bundles and/or fibrotic thickening of fat lobules (Figure 1). These areas did not represent scar fat because the patients had not undergone previous fat surgery. In agreement with these observations, the mean adipocyte size measured in patient cervical fat was lower than in control fat (ie, 1734, 1968, and 1692 μm2 for patients with LMNA mutations, HIV infection, or mtDNA mutations, respectively, versus 2988 μm2 for control subjects) (Figure 2A). In addition, a higher index of fibrosis was observed in fat from all patients as compared with control subjects (ie, 44.5, 25.6, and 17.5 for patients with LMNA mutations, HIV infection, or mtDNA mutations, respectively, versus 2.9 for control subjects) (Figure 2B). At the ultrastructural level, control cervical scAT demonstrated adipocytes with thin and regular cytoplasm and small intercellular areas. In fat from all patients, irregular cell outlines were observed, with thickened peripheral rims of cytoplasm and interstitial edema with compact and thick fibrils of collagen invading the intercellular area (Figure 3). In samples from control subjects and patients with LMNA or HIV-linked lipodystrophy, only rare macrophages were observed. Accordingly, in these cases, only a few cells were stained with CD68, a marker of proinflammatory macrophages (Figure 4A). In addition, mRNA expression of CD68 and labeling of the proinflammatory cytokines IL-6 and TNF-α was similar in cervical fat from patients with LMNA mutations or HIV infection and from control subjects (data not shown). Conversely, in the mtDNA-mutated lipomas, ultrastructural analysis revealed adipocytes with disrupted cell membranes, variably fragmented cytoplasmic rims, and large fat droplets lying free in the connective tissue, with numerous macrophages (Figure 3). Accordingly, in these samples, we observed many lipogranulomas, ie, CD68-positive macrophages organized into crown-like structures surrounding involutionary adipocytes (Figure 4, A and B), and CD68 mRNA expression was increased as compared with that in control samples (data not shown). In contrast to data previously reported in lipoatrophic areas from patients with LMNA mutations,20Araujo-Vilar D. Lattanzi G. Gonzalez-Mendez B. Costa-Freitas A.T. Prieto D. Columbaro M. Mattioli E. Victoria B. Martinez-Sanchez N. Ramazanova A. Fraga M. Beiras A. Forteza J. Dominguez-Gerpe L. Calvo C. Lado-Abeal J. Site-dependent differences in both prelamin A and adipogenic genes in subcutaneous adipose tissue of patients with type 2 familial partial lipodystrophy.J Med Genet. 2009; 46: 40-48Crossref PubMed Scopus (40) Google Scholar we did not detect ultrastructural alterations in the nucleus architecture of cervical adipocytes in the patients with LMNA mutations (data not shown). Considered together, these data demonstrate that the dystrophic phenotype of enlarged cervical adipose tissue is not associated with inflammatory features in patients with lipodystrophy with LMNA mutations or HIV infection. As shown in Figure 5, the surface occupied by blood vessels was decreased in patient samples as compared with control samples, as assessed using immunohistologic staining with the endothelial cells marker CD34. In addition, the surface occupied by small arteries, stained using α-SMA, was also decreased in all patient samples as compared with control samples (data not shown, and Figure 5B). The results were similar when we evaluated the number of vessels and small arteries in the adipose tissue areas excluding fibrosis (data not shown). The adipogenic transcription factors SREBP-1 and PPARγ revealed the same altered pattern of protein expression in patients with LMNA mutations or protease inhibitor–treated HIV infection: SREBP-1 was increased and PPARγ was underexpressed as compared with control samples (Figure 6). In buffalo humps from HIV-infected patients without current protease inhibitor treatment, expression of adipogenic genes was not significantly differ
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