1 |
Human lysyl hydroxylase isoforms:multifunctionality of human LH3 and the amino acids important for its collagen glycosyltransferase activitiesWang, C. (Chunguang) 17 September 2002 (has links)
Abstract
Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen.
Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types.
Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product.
The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.
|
2 |
Substrate specificity of lysyl hydroxylase isoforms and multifunctionality of lysyl hydroxylase 3Risteli, M. (Maija) 19 July 2008 (has links)
Abstract
Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated.
In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform.
Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule.
The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator.
|
3 |
Prolyl 4-hydroxylase:genomic cloning of the human and mouse α(II) subunit, tissue distribution of type I and II isoenzymes, and cloning and characterization of a novel prolyl 4-hydroxylase from Caenorhabditis elegansNissi, R. (Ritva) 04 July 2002 (has links)
Abstract
The collagens are a family of extracellular matrix proteins with a
widespread tissue distribution. Collagen biosynthesis requires the hydroxylation
of a number of proline residues by prolyl 4-hydroxylase. This posttranslational
modification is essential for the synthesis of all collagens, as 4-hydroxyproline
deficient collagens cannot form stable triple helices at body temperature.
The genes for the human and mouse prolyl 4-hydroxylase α(II) subunits
were cloned and characterized in this study. The human and mouse genes are 34.6
and 30.3 kb in size, respectively, consisting of 16 exons and 15 introns. The
intron sizes vary from 48-49 bp to over 8 kb in both genes. The 5' flanking
regions contain no TATA box, but there are several motifs that may act as
transcription factor binding sites. A novel mutually exclusively spliced exon 12a
was identified in both genes. Both variants of the α(II) subunit were found
to be expressed in a variety of tissues and both formed a fully active
recombinant tetramer with the β subunit when expressed in insect
cells.
Tissue distribution of the type I and type II prolyl 4-hydroxylase
isoenzymes was studied in developing, mature, and malignant cells and tissues by
immunofluorescence and Western blotting. The results indicate that the type I
isoenzyme is the main form in many cell types. Skeletal myocytes and smooth
muscle cells appeared to have the type I isoenzyme as their only prolyl
4-hydroxylase form, whereas the type II isoenzyme was clearly the main form in
chondrocytes. A strong signal for the type II enzyme was detected in cultured
umbilical and capillary endothelial cells, whereas the type I isoenzyme could not
be detected in these cells by immunostaining or Western blotting. Similar studies
on primary chondro- and osteosarcomas and benign bone tumours indicated that the
type I isoenzyme is the predominant form in both types of bone sarcoma, whereas
the type II isoenzyme was more abundantly expressed in benign tumours. In
chondrosarcomas, the type II isoenzyme was expressed in the nonmalignant
chondrocytes, whereas their malignant counterparts switched their expression
pattern to that of the type I isoenzyme.
Two isoforms of the catalytic prolyl 4-hydroxylase α subunit, PHY-1
and PHY-2, have previously been characterized from Caenorhabditis
elegans. This study reports the cloning and characterization of a
third C. elegans α subunit isoform, PHY-3, which is
much shorter than the previously characterized vertebrate and C.
elegans α subunits. Nematodes homozygous for a
phy-3 deletion were phenotypically wild type and fertile,
but the 4-hydroxyproline content of their early embryos was reduced by about 90%.
The expression of PHY-3 was found to be restricted to spermatheca of late larvae
and adult nematode, indicating that PHY-3 is likely to be involved in the
synthesis of collagens of the early embryo egg shells.
|
4 |
The desmoplastic response : mechanisms of tumour-induced fibrogenesisFearns, Colleen 03 May 2017 (has links)
The main concern of this thesis is with desmoplasia - a process in which excessive connective tissue is deposited in a neoplasm. This is a common phenomenon in neoplasia but one whose mechanisms are poorly understood. To study the process, I used a human malignant melanoma cell line (UCT-Mel 7) that was established in this laboratory and that, when injected into athymic mice, gave rise to tumours that showed a number of interesting features. Firstly, the tumour induced a marked desmoplastic response as evidenced by a high content of hydroxyproline in tumour lysates, intense staining for reticulin in sections of the tumour and infiltration of the tumour by host mesenchymal cells. Secondly, the desmoplasia was associated in UCT-Mel 7-derived tumours with an unusual phasic pattern of growth that was related to the in vitro passage number of the melanoma cells. On occasions, murine tumours developed at the site of inoculation of human tumour cells. I have identified 2 possible mechanisms by which UCT-Mel 7 cells could have induced the desmoplastic response: either the tumour cells could have exerted their effect indirectly, i.e. via macrophages, or they could have stimulated the host's stromal cells directly. UCT-Mel 7 cells were shown to be chemotactic for mouse macrophages and human foreskin fibroblasts were stimulated, in a dose-dependent manner, to synthesize increased amounts of collagen when co-cultured with mouse peritoneal exudate cells. Stimulation could only be effected by direct cell:cell contact; medium conditioned by macrophages was not effective. The amount of stimulation was not dependent on the state of activation of the peritoneal cells nor on the strain of mouse used. Tumour cells were also found to act directly. Co-culture of UCT-Mel 7 cells and fibroblasts resulted in increased collagen synthesis by the fibroblasts. Increased synthesis of the protein was reflected in an increase in the amount of collagen mRNA. UCT-Mel 7 cell stimulated in a dose-dependent manner with an absolute requirement for intimate cell:cell contact with the fibroblasts. DNA synthesis was not required. Dexamethasone, retinoic acid and the tumour promoter, phorbol myristate acetate, had significant primary effects on fibroblast collagen synthesis but did not modify the response to melanoma cells. Indomethacin, however, had a minimal primary effect upon the fibroblasts but significantly augmented the melanoma cell effect. The nature of the stimulatory cell:cell contact is still uncertain. The gap junction inhibitor, α-glycyrrhetinic acid, did not diminish the melanoma cell effect. Preliminary findings suggested that cell-surface proteoglycans may be important. Removal of the proteoglycans with the inhibitor of proteoglycan assembly, 4-methylumbelliferyl-β-D-xyloside, abrogated the melanoma cell:fibroblast interaction. Recombinant basic fibroblast growth factor did. not seem to be involved in the desmoplastic response. It was of incidental interest to note that this compound inhibited fibroblast collagen synthesis in a manner that was augmented by the concomitant addition of heparin. A surprising finding was the production of a potent inhibitor of collagen synthesis by superinduced cells of the mouse macrophage cell line, P388D₁. This inhibitor has not been fully characterised.
|
5 |
Análise ultraestrutural e implicação do RNA de interferência na fibrilogênese do colágeno V em pacientes com esclerodermia / Ultrastructural analysis and implication of RNA interference in collagen V fibrillogenesis in patients with sclerodermaMorais, Jymenez de 25 November 2014 (has links)
Introdução: Recentemente muitas são as funções atribuídas ao colágeno V em condições fisiológicas normais e em algumas doenças, como na esclerodermia. Esta proteína apresenta características estruturais singulares de regulação do diâmetro das fibrilas heterotípicas, e imunogenicidade, sendo capaz de desencadear uma resposta imune independente. Em descobertas recentes, desenvolvidas por nosso grupo, ficou evidenciado que o colágeno V apresenta histoarquitetura anômala, aumentando a sua imunoexpressão na pele e pulmão em estágios iniciais da doença, assim como, aumento do RNAm de suas cadeias, principalmente alfa2(V). Por esta razão e com o intuito de entender sobre os processos moleculares e ultraestruturais que correlacionam o COL V à fibrose na ES, a proposta do presente estudo foi realizar análise morfológica e ultraestrutural das cadeias, alfa1(V) e alfa2(V), da pele de pacientes com ES, assim como avaliar a influência do gene COL5A2 na fibrilogênese. Pacientes e Métodos: As análises foram desenvolvidas utilizando fragmento de pele obtidas de biópsia, tanto de pacientes como controles, sob aprovação do comitê de ética (CAPPesq 0331/10) e de acordo com Declaração de Helsinque. Participaram do estudo 7 pacientes (homens=3, mulheres=4) diagnosticados com ES há dois anos ou menos, e indivíduos voluntários saudáveis (n=7) utilizados como grupo controle. A biópsia das peles foi dividida para dois grupos de análises: estudos histológicos e cultura de fibroblastos; sob solução de formol a 10%, foram realizadas análises histomorfométricas [coloração de Hematoxilina e Eosina (H e E), Tricrômico de Masson, imunofluorescência (IM) para cadeias alfa1(V) e alfa2(V), e reconstrução tridimensional (Zeiss LSM 510 META/UV)], e em solução de glutaraldeído 2% para análise ultraestrutural (microscopia eletrônica de transmissão com imunomarcação com ouro coloidal). A quantificação das cadeias foi realizada por histomorfometria, através de análise de imagem utilizando o software Image Pro-Plus 6.0. Já a cultura de fibroblastos foi desenvolvida para avaliar: distribuição e perfil das cadeias alfa1(V) e alfa2(V) [reconstrução 3D (Zeiss LSM 510 META/UV)],expressão gênica COL5A1, COL5A2 e ITGA2 [qRT-PCR (Applied Biosystems)], e inibição temporária do gene COL5A2. Resultados: Na análise morfológica (H&E e Masson) observou-se modificação da histoarquitetura da pele dos pacientes, com espessamento e retificação da epiderme devido ao aumento na densidade das fibras colágenas, com projeções das papilas dérmicas em direção à derme papilar. Esse resultado foi confirmado na quantificação das cadeias (IM), evidenciado por perda acentuada da cadeia alfa1(V) na derme papilar [12,77 ± 1,344 vs 66,84± 3,36 (p < 0,0001)] e aumento significante da expressão da cadeias alfa1(V) e alfa2(V) na derme reticular alfa1(V) [7,657 ± 0,2133 vs 13,75 ± 0,958 ( p < 0,0001)] e alfa2(V) [5,072 ± 0,4117 vs 21,07 ± 0,790 (p=0,001)] dos pacientes quando comparados ao controle. Assim como visto na imunomarcação com ouro coloidal das cadeias do COLV, houve ausência de expressão linear da cadeia alfa1(V) na derme papilar, contrastando com a expressão da mesma na pele de controles, se contrapondo a forte imunoexpressão das cadeias alfa1(V) e alfa2(V) com uma maior evidência da cadeia alfa2(V) na região espessada pela junção lâmina basal e derme reticular. A reconstrução 3D em cultura de fibroblastos dérmicos demonstrou grande atividade das células de pacientes com ES, confirmado na expressão gênica dos COL5A1, COL5A2 e ITGA2, que se mostraram aumentados significantemente nos pacientes em relação ao controle. Por fim, após inibição do COL5A2 houve uma tendência ao aumento na expressão do COL5A1, e superexpressão da ITGA2. Conclusão: A alteração dérmica observada em pacientes com ES esta correlacionada com a modificação na distribuição das cadeias alfas do COLV, principalmente por perda acentuada da alfa1(V)3 homotrímera na derme papilar e superexpressão da alfa2(V) em capilares e vasos, interferindo na formação de matriz extracelular normal, sugerindo uma alteração pós traducional desta proteína, e que maiores estudos sobre a inibição da cadeia alfa2(V) são importantes para uma futura terapia gênica para atenuar os sintomas desencadeados nesta patologia / Introduction: Recently many functions are attributed to type V collagen in normal physiological conditions and in some diseases, such as scleroderma. This protein presents unique structural features for regulating the diameter of fibrils heterotypic and immunogenicity being capable of eliciting an immune response independent. In recent discoveries, developed by our group, it was evident that collagen V presents anomalous histoarchitecture, increasing the immunoexpression in the skin and lung in the early stages of the disease, as well as increased mRNA of his chains, mainly alpha2(V). For this reason and in order to understand the molecular and ultrastructural processes that correlate COL V fibrosis in SSc, the purpose of this study was to perform morphological and ultrastructural analysis of the chains alpha1(V) and alpha2(V) of the patient´s skin with ES, as well as to assess the influence of the COL5A2 gene in fibrillogenesis. Patients and Methods: The analyses were developed using skin fragment obtained from biopsy, both of patients and controls, under the approval of the ethics committee (CAPPesq 0331/10) and in accordance with the Declaration of Helsinki. The study included 7 patients (male = 3, female = 4) diagnosed with ES for two years or less, and healthy volunteers (n = 7) used as a control group. The biopsy of the skin was divided in two groups of analyzes: histological studies and cultured fibroblasts; Under formaldehyde solution 10%, histomorphometric analysis [staining with hematoxylin and eosin (H e E), Masson\'s trichrome, immunofluorescence (IM) to alpha1(V) and alpha2(V) chains, and three-dimensional reconstruction (Zeiss LSM 510 META were performed/UV), and in a solution of 2% glutaraldehyde for ultrastructural analysis (transmission electron microscopy with immunostaining with colloidal gold). Quantitation was performed by the chain histomorphometry by image analysis using Image Pro-Plus 6.0 software. Already a fibroblast culture was developed to assess: distribution and profile of the alpha1(V) and alpha2(V) chains, 3D reconstruction (Zeiss LSM 510 META / UV), gene expression of COL5A1, COL5A2 and ITGA2 [RT-qPCR (Applied Biosystems)], and temporary inhibition of the COL5A2 gene. Results: In the morphological analysis (H&E and Masson) was observed modification of histoarchitecture patient\'s skin, with thickening and rectification of the epidermis due to increased density of collagen fibers, with projections of dermal papillae toward the papillary dermis. This result was confirmed by quantification of the chains (IM), evidenced by marked loss of the alpha1(V) chain in the papillary dermis [12.77 ± 1.344 vs 66.84 ± 3.36 (p < 0.0001)] and significant increase in the expression of alpha1(V) and alpha2(V) chains, in the reticular dermis alpha1(V) chain [0.2133 ± 7.657 vs 0.958 ± 13.75 (p < 0.0001)] alfa2(V) chain [5.072 ± 0.4117 vs 21.07 ± 0.790 (p = 0.001)] of patients when compared to control. As seen in the immunostaining with colloidal gold chains of COLV, there was no linear expression of the alpha1(V) chain in the papillary dermis, contrasting with the same expression on the skin of controls, in contrast to strong immunoexpression of alpha1(V) and alpha2(V) chains, with a higher evidence of alpha2(V) chain in the junction region by a thickened basement membrane and reticular dermis. The 3D reconstruction of dermal fibroblasts in culture demonstrated large cell activity of patients with SSc, confirmed the gene expression of COL5A1, COL5A2 and ITGA2, which showed significantly increased in patients compared to control. Finally, after inhibition of COL5A2 there was a trend to increase in the expression of the COL5A1, and overexpression of ITGA2. Conclusion: The dermal alteration observed in SSc patients is correlated with the change in the distribution of the collagen alpha (V) chains, mainly by marked loss of alpha1(V)3 homotrimer the papillary dermis and overexpression of the alpha2(V) in capillaries and vessels, interfering with the normal extracellular matrix formation, suggesting a post-translational modification of this protein, and further studies on the inhibition of chain alpha2(V) are important for future gene therapy to attenuate the symptoms of this pathology triggered
|
6 |
Análise ultraestrutural e implicação do RNA de interferência na fibrilogênese do colágeno V em pacientes com esclerodermia / Ultrastructural analysis and implication of RNA interference in collagen V fibrillogenesis in patients with sclerodermaJymenez de Morais 25 November 2014 (has links)
Introdução: Recentemente muitas são as funções atribuídas ao colágeno V em condições fisiológicas normais e em algumas doenças, como na esclerodermia. Esta proteína apresenta características estruturais singulares de regulação do diâmetro das fibrilas heterotípicas, e imunogenicidade, sendo capaz de desencadear uma resposta imune independente. Em descobertas recentes, desenvolvidas por nosso grupo, ficou evidenciado que o colágeno V apresenta histoarquitetura anômala, aumentando a sua imunoexpressão na pele e pulmão em estágios iniciais da doença, assim como, aumento do RNAm de suas cadeias, principalmente alfa2(V). Por esta razão e com o intuito de entender sobre os processos moleculares e ultraestruturais que correlacionam o COL V à fibrose na ES, a proposta do presente estudo foi realizar análise morfológica e ultraestrutural das cadeias, alfa1(V) e alfa2(V), da pele de pacientes com ES, assim como avaliar a influência do gene COL5A2 na fibrilogênese. Pacientes e Métodos: As análises foram desenvolvidas utilizando fragmento de pele obtidas de biópsia, tanto de pacientes como controles, sob aprovação do comitê de ética (CAPPesq 0331/10) e de acordo com Declaração de Helsinque. Participaram do estudo 7 pacientes (homens=3, mulheres=4) diagnosticados com ES há dois anos ou menos, e indivíduos voluntários saudáveis (n=7) utilizados como grupo controle. A biópsia das peles foi dividida para dois grupos de análises: estudos histológicos e cultura de fibroblastos; sob solução de formol a 10%, foram realizadas análises histomorfométricas [coloração de Hematoxilina e Eosina (H e E), Tricrômico de Masson, imunofluorescência (IM) para cadeias alfa1(V) e alfa2(V), e reconstrução tridimensional (Zeiss LSM 510 META/UV)], e em solução de glutaraldeído 2% para análise ultraestrutural (microscopia eletrônica de transmissão com imunomarcação com ouro coloidal). A quantificação das cadeias foi realizada por histomorfometria, através de análise de imagem utilizando o software Image Pro-Plus 6.0. Já a cultura de fibroblastos foi desenvolvida para avaliar: distribuição e perfil das cadeias alfa1(V) e alfa2(V) [reconstrução 3D (Zeiss LSM 510 META/UV)],expressão gênica COL5A1, COL5A2 e ITGA2 [qRT-PCR (Applied Biosystems)], e inibição temporária do gene COL5A2. Resultados: Na análise morfológica (H&E e Masson) observou-se modificação da histoarquitetura da pele dos pacientes, com espessamento e retificação da epiderme devido ao aumento na densidade das fibras colágenas, com projeções das papilas dérmicas em direção à derme papilar. Esse resultado foi confirmado na quantificação das cadeias (IM), evidenciado por perda acentuada da cadeia alfa1(V) na derme papilar [12,77 ± 1,344 vs 66,84± 3,36 (p < 0,0001)] e aumento significante da expressão da cadeias alfa1(V) e alfa2(V) na derme reticular alfa1(V) [7,657 ± 0,2133 vs 13,75 ± 0,958 ( p < 0,0001)] e alfa2(V) [5,072 ± 0,4117 vs 21,07 ± 0,790 (p=0,001)] dos pacientes quando comparados ao controle. Assim como visto na imunomarcação com ouro coloidal das cadeias do COLV, houve ausência de expressão linear da cadeia alfa1(V) na derme papilar, contrastando com a expressão da mesma na pele de controles, se contrapondo a forte imunoexpressão das cadeias alfa1(V) e alfa2(V) com uma maior evidência da cadeia alfa2(V) na região espessada pela junção lâmina basal e derme reticular. A reconstrução 3D em cultura de fibroblastos dérmicos demonstrou grande atividade das células de pacientes com ES, confirmado na expressão gênica dos COL5A1, COL5A2 e ITGA2, que se mostraram aumentados significantemente nos pacientes em relação ao controle. Por fim, após inibição do COL5A2 houve uma tendência ao aumento na expressão do COL5A1, e superexpressão da ITGA2. Conclusão: A alteração dérmica observada em pacientes com ES esta correlacionada com a modificação na distribuição das cadeias alfas do COLV, principalmente por perda acentuada da alfa1(V)3 homotrímera na derme papilar e superexpressão da alfa2(V) em capilares e vasos, interferindo na formação de matriz extracelular normal, sugerindo uma alteração pós traducional desta proteína, e que maiores estudos sobre a inibição da cadeia alfa2(V) são importantes para uma futura terapia gênica para atenuar os sintomas desencadeados nesta patologia / Introduction: Recently many functions are attributed to type V collagen in normal physiological conditions and in some diseases, such as scleroderma. This protein presents unique structural features for regulating the diameter of fibrils heterotypic and immunogenicity being capable of eliciting an immune response independent. In recent discoveries, developed by our group, it was evident that collagen V presents anomalous histoarchitecture, increasing the immunoexpression in the skin and lung in the early stages of the disease, as well as increased mRNA of his chains, mainly alpha2(V). For this reason and in order to understand the molecular and ultrastructural processes that correlate COL V fibrosis in SSc, the purpose of this study was to perform morphological and ultrastructural analysis of the chains alpha1(V) and alpha2(V) of the patient´s skin with ES, as well as to assess the influence of the COL5A2 gene in fibrillogenesis. Patients and Methods: The analyses were developed using skin fragment obtained from biopsy, both of patients and controls, under the approval of the ethics committee (CAPPesq 0331/10) and in accordance with the Declaration of Helsinki. The study included 7 patients (male = 3, female = 4) diagnosed with ES for two years or less, and healthy volunteers (n = 7) used as a control group. The biopsy of the skin was divided in two groups of analyzes: histological studies and cultured fibroblasts; Under formaldehyde solution 10%, histomorphometric analysis [staining with hematoxylin and eosin (H e E), Masson\'s trichrome, immunofluorescence (IM) to alpha1(V) and alpha2(V) chains, and three-dimensional reconstruction (Zeiss LSM 510 META were performed/UV), and in a solution of 2% glutaraldehyde for ultrastructural analysis (transmission electron microscopy with immunostaining with colloidal gold). Quantitation was performed by the chain histomorphometry by image analysis using Image Pro-Plus 6.0 software. Already a fibroblast culture was developed to assess: distribution and profile of the alpha1(V) and alpha2(V) chains, 3D reconstruction (Zeiss LSM 510 META / UV), gene expression of COL5A1, COL5A2 and ITGA2 [RT-qPCR (Applied Biosystems)], and temporary inhibition of the COL5A2 gene. Results: In the morphological analysis (H&E and Masson) was observed modification of histoarchitecture patient\'s skin, with thickening and rectification of the epidermis due to increased density of collagen fibers, with projections of dermal papillae toward the papillary dermis. This result was confirmed by quantification of the chains (IM), evidenced by marked loss of the alpha1(V) chain in the papillary dermis [12.77 ± 1.344 vs 66.84 ± 3.36 (p < 0.0001)] and significant increase in the expression of alpha1(V) and alpha2(V) chains, in the reticular dermis alpha1(V) chain [0.2133 ± 7.657 vs 0.958 ± 13.75 (p < 0.0001)] alfa2(V) chain [5.072 ± 0.4117 vs 21.07 ± 0.790 (p = 0.001)] of patients when compared to control. As seen in the immunostaining with colloidal gold chains of COLV, there was no linear expression of the alpha1(V) chain in the papillary dermis, contrasting with the same expression on the skin of controls, in contrast to strong immunoexpression of alpha1(V) and alpha2(V) chains, with a higher evidence of alpha2(V) chain in the junction region by a thickened basement membrane and reticular dermis. The 3D reconstruction of dermal fibroblasts in culture demonstrated large cell activity of patients with SSc, confirmed the gene expression of COL5A1, COL5A2 and ITGA2, which showed significantly increased in patients compared to control. Finally, after inhibition of COL5A2 there was a trend to increase in the expression of the COL5A1, and overexpression of ITGA2. Conclusion: The dermal alteration observed in SSc patients is correlated with the change in the distribution of the collagen alpha (V) chains, mainly by marked loss of alpha1(V)3 homotrimer the papillary dermis and overexpression of the alpha2(V) in capillaries and vessels, interfering with the normal extracellular matrix formation, suggesting a post-translational modification of this protein, and further studies on the inhibition of chain alpha2(V) are important for future gene therapy to attenuate the symptoms of this pathology triggered
|
Page generated in 0.1037 seconds