RESUMO
The introduction of combination antiretroviral therapy (cART) led to substantial improvement in mortality and morbidity of HIV-1 infection. However, the poor penetration of antiretroviral agents to HIV-1 reservoirs limit the ability of the antiretroviral agents to eliminate the virus. Mesenteric lymph nodes (MLNs) are one of the main HIV-1 reservoirs in patients under suppressive cART. Intestinal lymphatic absorption pathway substantially increases the concentration of lipophilic drugs in mesenteric lymph and MLNs when they are co-administered with long-chain triglyceride (LCT). Chylomicrons (CM) play a crucial role in the intestinal lymphatic absorption as they transport drugs to the lymph lacteals rather than blood capillary by forming CM-drug complexes in the enterocytes. Thus, lipophilic antiretroviral drugs could potentially be delivered to HIV-1 reservoirs in MLNs by LCT-based formulation approach. In this study, protease inhibitors (PIs) were initially screened for their potential for intestinal lymphatic targeting using a computational model. The candidates were further assessed for their experimental affinity to CM. Tipranavir (TPV) was the only-candidate with substantial affinity to both artificial and natural CM in vitro and ex vivo. Pharmacokinetics and biodistribution studies were then performed to evaluate the oral bioavailability and intestinal lymphatic targeting of TPV in rats. The results showed similar oral bioavailability of TPV with and without co-administration of LCT vehicle. Although LCT-based formulation led to 3-fold higher concentrations of TPV in mesenteric lymph compared to plasma, the levels of the drug in MLNs were similar to plasma in both LCT-based and lipid-free formulation groups. Thus, LCT-based formulation approach alone was not sufficient for effective delivery of TPV to MLNs. Future efforts should be directed to a combined highly lipophilic prodrugs/lipid-based formulation approach to target TPV, other PIs and potentially other classes of antiretroviral agents to viral reservoirs within the mesenteric lymphatic system.
Assuntos
HIV-1 , Administração Oral , Animais , Humanos , Linfonodos/metabolismo , Piridinas , Pironas , Ratos , Sulfonamidas , Distribuição Tecidual , TriglicerídeosRESUMO
[Figure: see text].
Assuntos
Aterosclerose/enzimologia , Colesterol/metabolismo , Células Espumosas/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Esterol Esterase/metabolismo , Animais , Aorta/enzimologia , Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Modelos Animais de Doenças , Células Espumosas/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Células RAW 264.7RESUMO
OBJECTIVE: To explore the role of LAL (lysosomal acid lipase) in macrophage cholesterol efflux and whole-body reverse cholesterol transport. APPROACH AND RESULTS: Immortalized peritoneal macrophages from lal-/- mice showed reduced expression of ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1), reduced production of the regulatory oxysterol 27-hydroxycholesterol, and impaired suppression of cholesterol synthesis on exposure to acetylated low-density lipoprotein when compared with lal+/+ macrophages. LAL-deficient mice also showed reduced hepatic ABCG5 (ATP-binding cassette transporter G5) and ABCG8 (ATP-binding cassette transporter G8) expression compared with lal+/+ mice. LAL-deficient macrophages loaded with [3H]-cholesteryl oleate-labeled acetylated low-density lipoprotein showed impaired efflux of released [3H]-cholesterol to apoA-I (apolipoprotein A-I), with normalization of [3H]-cholesteryl ester levels and partial correction of ABCA1 expression and cholesterol efflux to apoA-I when treated with exogenous rhLAL (recombinant human LAL protein). LAL-deficient mice injected intraperitoneally with lal-/- macrophages cholesterol loaded and labeled in the same way exhibited only 1.55±0.35% total injected [3H]-cholesterol counts appearing in the feces for 48 h (n=30), compared with 5.38±0.92% in lal+/+ mice injected with labeled lal+/+ macrophages (n=27), P<0.001. To mimic the therapeutic condition of delivery of supplemental LAL in vivo, injection of labeled lal-/- macrophages into lal+/+ mice resulted in a significant increase in reverse cholesterol transport (2.60±0.46% of 3H-cholesterol counts in feces at 48 hours [n=19]; P<0.001 when compared with injection into lal-/- mice). CONCLUSIONS: These results indicate a critical role for LAL in promoting both macrophage and whole-body reverse cholesterol transport and the ability of supplemental LAL to be taken up and correct reverse cholesterol transport in vivo.
Assuntos
Colesterol/metabolismo , Macrófagos Peritoneais/enzimologia , Esterol Esterase/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Transporte Biológico , Linhagem Celular , Colesterol/sangue , Fezes/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Fígado/metabolismo , Camundongos da Linhagem 129 , Camundongos Knockout , Esterol Esterase/deficiência , Esterol Esterase/genéticaRESUMO
A low level of HDL cholesterol (HDL-C) is a common clinical scenario and an important marker for increased cardiovascular risk. Many patients with very low or very high HDL-C have a rare mutation in one of several genes, but identification of the molecular abnormality in patients with extreme HDL-C is rarely performed in clinical practice. We investigated the accuracy and diagnostic yield of a targeted next-generation sequencing (NGS) assay for extreme levels of HDL-C. We developed a targeted NGS panel to capture the exons, intron/exon boundaries, and untranslated regions of 26 genes with highly penetrant effects on plasma lipid levels. We sequenced 141 patients with extreme HDL-C levels and prioritized variants in accordance with medical genetics guidelines. We identified 35 pathogenic and probably pathogenic variants in HDL genes, including 21 novel variants, and performed functional validation on a subset of these. Overall, a molecular diagnosis was established in 35.9% of patients with low HDL-C and 5.2% with high HDL-C, and all prioritized variants identified by NGS were confirmed by Sanger sequencing. Our results suggest that a molecular diagnosis can be identified in a substantial proportion of patients with low HDL-C using targeted NGS.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Doenças Cardiovasculares/genética , HDL-Colesterol/sangue , HDL-Colesterol/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Transportador 1 de Cassete de Ligação de ATP/sangue , Alelos , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Éxons , Feminino , Estudos de Associação Genética , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
BACKGROUND: ATP-binding cassette transporter A1 (ABCA1) mediates the lipidation of exchangeable apolipoproteins, the rate-limiting step in the formation of high density lipoproteins (HDL). We previously demonstrated that HDL oxidized ex vivo by peroxidase-generated tyrosyl radical (tyrosylated HDL, tyrHDL) increases the availability of cellular cholesterol for efflux and reduces the development of atherosclerosis when administered to apolipoprotein E-deficient mice as compared to treatment with control HDL. RESULTS: In the current study we determined that tyrHDL requires functional ABCA1 for this enhanced activity. Like lipid-free apolipoprotein A-I (apoA-I), tyrHDL increases total and cell surface ABCA1, inhibits calpain-dependent and -independent proteolysis of ABCA1, and can be bound by cell surface ABCA1 in human skin fibroblasts. Additionally, tyrHDL apoproteins are susceptible to digestion by enteropeptidase like lipid-free apoA-I, but unlike lipid-bound apoA-I on HDL, which is resistant to proteolysis. CONCLUSIONS: These results provide the first evidence that lipid-bound apolipoproteins on the surface of spherical HDL particles can behave like lipid-free apoA-I to increase ABCA1 protein levels and activity.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Lipoproteínas HDL/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Apolipoproteína A-I/química , Calpaína/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Enteropeptidase/química , Esterificação , Expressão Gênica , Humanos , Lipoproteínas HDL/química , Oxirredução , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Proteólise , Tirosina/químicaRESUMO
Lipid A, the active moiety of LPS, exerts its effects through interaction with TLR4, triggering a signalling cascade that results in the release of pro-inflammatory cytokines. Eritoran is a lipid A analogue that competes with LPS for binding to TLR4; however, after intravenous administration, it undergoes a time-dependent deactivation as a consequence of binding to high-density lipoproteins (HDLs). The site of eritoran association with HDL remains unknown. Therefore the aim of this study was to determine if HDL-associated apolipoproteins A1, A2, serum amyloid A (SAA) and C1, inhibit the ability of eritoran to block LPS-induced TNF-α release from whole blood. Eritoran activity after LPS stimulation in human whole blood was assessed in the presence of reconstituted HDL (rHDL) containing different apos. In rHDL, the major apolipoproteins in both the healthy and septic state, A1 and SAA, caused a significant reduction in eritoran antagonistic activity and had a greater effect than minor apolipoproteins A2 and C1. Apolipoproteins associated with HDL are likely to facilitate eritoran deactivation. Apolipoproteins A1 and SAA should be of particular focus as they are the major apos found on HDL in both the healthy and septic state. Further evaluation of the physical association between apolipoproteins and eritoran should be explored.
Assuntos
Células Sanguíneas/efeitos dos fármacos , Dissacarídeos/antagonistas & inibidores , Lipídeo A/antagonistas & inibidores , Lipoproteínas HDL/imunologia , Fosfatos Açúcares/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Ligação Competitiva , Células Sanguíneas/imunologia , Células Sanguíneas/metabolismo , Células Sanguíneas/patologia , Dissacarídeos/farmacologia , Humanos , Estrutura Molecular , Proteína Amiloide A Sérica/metabolismo , Fosfatos Açúcares/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
ATP-binding cassette transporter A1 (ABCA1) mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, and its expression is regulated primarily by oxysterol-dependent activation of liver X receptors. We previously reported that ABCA1 expression and HDL formation are impaired in the lysosomal cholesterol storage disorder Niemann-Pick disease type C1 and that plasma HDL-C is low in the majority of Niemann-Pick disease type C patients. Here, we show that ABCA1 regulation and activity are also impaired in cholesteryl ester storage disease (CESD), caused by mutations in the LIPA gene that result in less than 5% of normal lysosomal acid lipase (LAL) activity. Fibroblasts from patients with CESD showed impaired up-regulation of ABCA1 in response to low density lipoprotein (LDL) loading, reduced phospholipid and cholesterol efflux to apolipoprotein A-I, and reduced α-HDL particle formation. Treatment of normal fibroblasts with chloroquine to inhibit LAL activity reduced ABCA1 expression and activity, similar to that of CESD cells. Liver X receptor agonist treatment of CESD cells corrected ABCA1 expression but failed to correct LDL cholesteryl ester hydrolysis and cholesterol efflux to apoA-I. LDL-induced production of 27-hydroxycholesterol was reduced in CESD compared with normal fibroblasts. Treatment with conditioned medium containing LAL from normal fibroblasts or with recombinant human LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation, and production of 27-hydroxycholesterol by CESD cells. These results provide further evidence that the rate of release of cholesterol from late endosomes/lysosomes is a critical regulator of ABCA1 expression and activity, and an explanation for the hypoalphalipoproteinemia seen in CESD patients.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Doença do Armazenamento de Colesterol Éster/genética , Lipoproteínas HDL/metabolismo , Doença de Wolman/genética , Transportador 1 de Cassete de Ligação de ATP , Cloroquina/metabolismo , Colesterol/metabolismo , Doença do Armazenamento de Colesterol Éster/metabolismo , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/metabolismo , Humanos , Lipoproteínas/metabolismo , Mutação , Fosfolipídeos/metabolismo , Proteínas Recombinantes/metabolismo , Pele/metabolismo , Esteróis/química , Doença de Wolman/metabolismo , Doença de WolmanRESUMO
The Niemann-Pick C1 and C2 (NPC1 and NPC2) proteins have a central role in regulating the transport of lipoprotein-derived cholesterol from endocytic compartments to the endoplasmic reticulum for esterification by acyl-CoA:cholesterol acyltransferase (ACAT) and feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway. Since the NPC1 gene/protein has recently been shown to be downregulated by feedback inhibition of the SREBP pathway, the present study was performed to determine whether physiological downregulation of the NPC1 gene/protein alters the transport and metabolism of low-density lipoprotein (LDL)-derived cholesterol in human fibroblasts. To perform this study, three different culture conditions were used that included fibroblasts grown in lipoprotein-deficient serum (LPDS), LPDS supplemented with LDL, and LPDS supplemented with LDL, followed by equilibration in the absence of LDL to allow the transport of LDL-derived cholesterol from endocytic compartments and equilibration of cellular sterol pools. The results from this study indicated that in addition to the NPC1 gene/protein, the NPC2 gene/protein was also downregulated by LDL-derived cholesterol-dependent feedback inhibition and that downregulation of both the NPC1 and NPC2 genes/proteins was associated with the sequestration of LDL-derived cholesterol within endocytic compartments, including late endosomes/lysosomes after equilibration. Therefore, it is proposed that physiological and coordinate downregulation of the NPC1 and NPC2 genes/proteins promotes the sequestration of LDL-derived cholesterol within endocytic compartments and serves a role in maintaining intracellular cholesterol homeostasis.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , LDL-Colesterol/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Linhagem Celular , Regulação para Baixo , Endossomos/metabolismo , Fibroblastos/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Proteína C1 de Niemann-Pick , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Proteínas de Transporte VesicularRESUMO
BACKGROUND: Accumulation of excess cholesterol by intimal arterial smooth muscle cells (SMCs) contributes to the formation of foam cells in atherosclerotic lesions. The purpose of this study was to examine the expression and activity of ATP-binding cassette transporter A1 (ABCA1) in model intimal and medial arterial SMCs, in human atherosclerotic coronary artery intimal and medial layers, and in human intimal and medial SMCs. METHODS AND RESULTS: Model intimal arterial SMCs showed increased cholesteryl ester accumulation, absence of apolipoprotein A-I-mediated lipid efflux, markedly diminished ABCA1 expression, and poor apoA-I binding compared with medial-layer SMCs. Total ABCA1 mRNA and SMC-specific ABCA1 protein levels were diminished in the intimal layer compared with the medial layer of atherosclerotic human coronary arteries. Increased expression of ABCA1 by liver X receptor agonist treatment or gene transfection failed to correct apolipoprotein A-I binding, lipid efflux, or high-density lipoprotein particle formation by intima-type SMCs. In addition to impaired ABCA1 expression, intima-type SMCs appear to lack a critical binding factor or factors required for the apolipoprotein A-I-ABCA1 interaction, cholesterol efflux, and high-density lipoprotein particle formation. CONCLUSIONS: ABCA1 expression is reduced in cultured model intimal and human atherosclerotic lesion SMCs, suggesting that reduced ABCA1 activity contributes to smooth muscle foam cell formation in the intima.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerose/metabolismo , Músculo Liso Vascular/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Aorta Torácica/citologia , Apolipoproteína A-I/farmacologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Linhagem Celular , Colesterol/metabolismo , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/fisiopatologia , Vasos Coronários/patologia , Células Espumosas/patologia , Humanos , Lipoproteínas HDL/metabolismo , Músculo Liso Vascular/patologia , Fenótipo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos WKY , Túnica Íntima/metabolismo , Túnica Íntima/patologiaRESUMO
Impaired cell cholesterol trafficking in Niemann-Pick type C (NPC) disease results in the first known instance of impaired regulation of the ATP-binding cassette transporter A1 (ABCA1), a lipid transporter mediating the rate-limiting step in high density lipoprotein (HDL) formation, as a cause of low plasma HDL-cholesterol in humans. We show here that treatment of human NPC1(-/-) fibroblasts with the liver X receptor (LXR) agonist TO-901317 increases ABCA1 expression and activity in human NPC1(-/-) fibroblasts, as indicated by near normalization of efflux of radiolabeled phosphatidylcholine and a marked increase in efflux of cholesterol mass to apoA-I. LXR agonist treatment prior to and during apoA-I incubation resulted in reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes, as well as cholesterol mass, in NPC1(-/-) cells. HDL species in human NPC disease plasma showed the same pattern of diminished large, cholesterol-rich alpha-1 HDL particles as seen in isolated heterozygous ABCA1 deficiency. Incubating NPC1(-/-) fibroblasts with the LXR agonist normalized the pattern of HDL particle formation by these cells. ABCG1, another LXR target gene involved in cholesterol efflux to HDL, also showed diminished expression in NPC1(-/-) fibroblasts and increased expression upon LXR agonist treatment. These results suggest that NPC1 mutations can be largely bypassed and that NPC1 protein function is non-essential for the trafficking and removal of cellular cholesterol if the down-stream defects in ABCA1 and ABCG1 regulation in NPC disease cells are corrected using an LXR agonist.
Assuntos
Apolipoproteína A-I/fisiologia , Fibroblastos/metabolismo , Lipídeos/química , Lipoproteínas HDL/química , Doenças de Niemann-Pick/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Trifosfato de Adenosina/química , Apolipoproteína A-I/metabolismo , Western Blotting , Proteínas de Transporte/fisiologia , Linhagem Celular , Células Cultivadas , Colesterol/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas/química , Glicoproteínas de Membrana/fisiologia , Mutação , Proteína C1 de Niemann-Pick , Fosfatidilcolinas/químicaRESUMO
High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Hepatócitos/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas VLDL/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/farmacologia , Apolipoproteínas B/metabolismo , Células Cultivadas , Feminino , Hepatócitos/citologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-DawleyRESUMO
The cholesterol trafficking defect in Niemann-Pick type C (NPC) disease leads to impaired regulation of cholesterol esterification, cholesterol synthesis, and low density lipoprotein receptor activity. The ATP-binding cassette transporter A1 (ABCA1), which mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, is also regulated by cell cholesterol content. To determine whether the Niemann-Pick C1 protein alters the expression and activity of ABCA1, we determined the ability of apolipoprotein A-I (apoA-I) to deplete pools of cellular cholesterol and phospholipids in human fibroblasts derived from NPC1+/+, NPC1+/-, and NPC1-/- subjects. Efflux of low density lipoprotein-derived, non-lipoprotein, plasma membrane, and newly synthesized pools of cell cholesterol by apoA-I was diminished in NPC1-/- cells, as was efflux of phosphatidylcholine and sphingomyelin. NPC1+/- cells showed intermediate levels of lipid efflux compared with NPC1+/+ and NPC1-/- cells. Binding of apoA-I to cholesterol-loaded and non-cholesterol-loaded cells was highest for NPC1+/- cells, with NPC1+/+ and NPC1-/- cells showing similar levels of binding. ABCA1 mRNA and protein levels increased in response to cholesterol loading in NPC1+/+ and NPC1+/- cells but showed low levels at base line and in response to cholesterol loading in NPC1-/- cells. Consistent with impaired ABCA1-dependent lipid mobilization to apoA-I for HDL particle formation, we demonstrate for the first time decreased plasma HDL-cholesterol levels in 17 of 21 (81%) NPC1-/- subjects studied. These results indicate that the cholesterol trafficking defect in NPC disease results in reduced activity of ABCA1, which we suggest is responsible for the low HDL-cholesterol in the majority of NPC subjects and partially responsible for the overaccumulation of cellular lipids in this disorder.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Metabolismo dos Lipídeos , Doenças de Niemann-Pick/metabolismo , Doença de Tangier/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adolescente , Adulto , Apolipoproteína A-I/metabolismo , Northern Blotting , Western Blotting , Criança , Pré-Escolar , Colesterol/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Lipoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Mutação , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Sexuais , Esfingomielinas/metabolismo , Fatores de TempoRESUMO
Transforming growth factor-beta1 is a fibrogenic cytokine that is important in the development of fibroproliferative disorders of the skin after injury. To investigate the role of transforming growth factor-beta1 produced by keratinocytes during wound healing, a plasmid with the human transforming growth factor-beta1 gene coupled with the keratin 14 promoter (pG3Z: K14-TGF-beta1) was constructed. The construct was tested successfully in vitro before being used to generate transgenic animals, which were subsequently bred into homozygous and heterozygous lines. Genotype screening of founders and progeny was performed by Southern blotting and targeting of the transgene to the epidermis by the keratin 14 promoter was shown by reverse transcription polymerase chain reaction. The major phenotypic change observed in the transgenic animals was "scruffiness" of the fur attributed to transgene expression in the skin, seen primarily in the homozygous line. A significant reduction in the rate of reepithelialization of full-thickness excisional wounds of dorsal skin was seen in homozygous animals compared with normal litter-mate controls at day 7 (p < 0.05, Fisher's Exact test) and day 9 (p < 0.01) postwounding. Wounds in heterozygous animals also healed more slowly at day 9 (p < 0.01). Northern analysis of mRNA extracted from the wounds showed increased human transforming growth factor-beta1 message levels in homozygous and heterozygous animals, maximal at day 5. Significant increases in transforming growth factor-beta1 activity in healing wounds measured using the plasminogen activator inhibitor-1/luciferase assay were found in the transgenic strains at day 9 postinjury as compared with the normal litter-mate control mice (p < 0.001, ANOVA). Type I procollagen mRNA expression was higher in the homozygous and heterozygous animals, with the highest levels reached at day 9. By day 5 postwounding, biopsies of both homozygous and heterozygous tissues were significantly higher in collagen as compared with wounds in control animals (p < 0.05, ANOVA). Based on these data, the K14-TGF-beta1 transgenic mouse shows that excessive latent transforming growth factor-beta1 produced in the epidermal layer of the skin delays reepithelialization in excisional wounds but subsequently the cells of the epidermis stimulate dermal fibroblasts leading to fibrosis through a paracrine mechanism.