RESUMO
Numerous studies have demonstrated that endostatin (ES), a potent angiostatic peptide derived from collagen type XVIII α 1 chain and encoded by COL18A1, is elevated in pulmonary arterial hypertension (PAH). It is important to note that elevated ES has consistently been associated with altered hemodynamics, poor functional status, and adverse outcomes in adult and pediatric PAH. This study used serum samples from patients with Group I PAH and plasma and tissue samples derived from the Sugen/hypoxia rat pulmonary hypertension model to define associations between COL18A1/ES and disease development, including hemodynamics, right ventricle (RV) remodeling, and RV dysfunction. Using cardiac magnetic resonance imaging and advanced hemodynamic assessments with pressure-volume loops in patients with PAH to assess RV-pulmonary arterial coupling, we observed a strong relationship between circulating ES levels and metrics of RV structure and function. Specifically, RV mass and the ventricular mass index were positively associated with ES, whereas RV ejection fraction and RV-pulmonary arterial coupling were inversely associated with ES levels. Our animal data demonstrate that the development of pulmonary hypertension is associated with increased COL18A1/ES in the heart as well as the lungs. Disease-associated increases in COL18A1 mRNA and protein were most pronounced in the RV compared with the left ventricle and lung. COL18A1 expression in the RV was strongly associated with disease-associated changes in RV mass, fibrosis, and myocardial capillary density. These findings indicate that COL18A1/ES increases early in disease development in the RV and implicates COL18A1/ES in pathologic RV dysfunction in PAH.
Assuntos
Endostatinas , Disfunção Ventricular Direita , Remodelação Ventricular , Animais , Endostatinas/metabolismo , Humanos , Masculino , Feminino , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/fisiopatologia , Ratos , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/fisiopatologia , Hipertensão Arterial Pulmonar/patologia , Ratos Sprague-Dawley , Colágeno Tipo XVIII/metabolismo , Colágeno Tipo XVIII/genética , Pessoa de Meia-Idade , Adulto , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/patologia , Progressão da Doença , Modelos Animais de Doenças , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Ventrículos do Coração/patologiaRESUMO
For over a decade, diabetic neuropathy has exhibited great emergence in diabetic patients. Though there are numerous impediments in understanding the underlying pathology it is not that enough to conclude. Initially, there was no intricate protocol for diagnosis as its symptoms mimic most of the neurodegenerative disorders and demyelinating diseases. Continuous research on this, reveals many pathological correlates which are also detectable clinically. The most important pathologic manifestation is imbalanced angiogenesis/neo-vascularization. This review is completely focused on established pathogenesis and anti-angiogenic agents which are physiological signal molecules by the origin. Those agents can also be used externally to inhibit those pathogenic pathways. Pathologically DN demonstrates the misbalanced expression of many knotty factors like VEGF, FGF2, TGFb, NF-kb, TNF-a, MMP, TIMP, and many minor factors. Their pathway towards the incidence of DN is quite interrelated. Many anti-angiogenic agents inhibit neovascularization to many extents, but out of them predominantly inhibition of angiogenic activity is shared by endostatin which is now in clinical trial phase II. It inhibits almost all angiogenic factors and it is possible because they share interrelated pathogenesis towards imbalanced angiogenesis. Endostatin is a physiological signal molecule produced by the proteolytic cleavage of collagen XVIII. It has also a broad research profile in the field of medical research and further investigation can show promising therapeutic effects for benefit of mankind.
Assuntos
Colágeno Tipo XVIII/metabolismo , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/metabolismo , Endostatinas/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Inibidores da Angiogênese , Colágeno Tipo XVIII/farmacologia , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Neuropatias Diabéticas/complicações , Neuropatias Diabéticas/genética , Endostatinas/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Redes e Vias Metabólicas/genética , Neovascularização Fisiológica/genéticaRESUMO
Pulmonary arterial hypertension (PAH) is an incurable disease characterized by disordered and dysfunctional angiogenesis leading to small-vessel loss and an obliterative vasculopathy. The pathogenesis of PAH is not fully understood, but multiple studies have demonstrated links between elevated angiostatic factors, disease severity, and adverse clinical outcomes. ES (endostatin), one such circulating angiostatic peptide, is the cleavage product of the proteoglycan COL18A1 (collagen α1[XVIII] chain). Elevated serum ES is associated with increased mortality and disease severity in PAH. A nonsynonymous variant of ES (aspartic acid-to-asparagine substitution at amino acid 104; p.D104N) is associated with differences in PAH survival. Although COL18A1/ES expression is markedly increased in remodeled pulmonary vessels in PAH, the impact of ES on pulmonary endothelial cell (PEC) biology and molecular contributions to PAH severity remain undetermined. In the present study, we characterized the effects of exogenous ES on human PEC biology and signaling. We demonstrated that ES inhibits PEC migration, proliferation, and cell survival, with significant differences between human variants, indicating that they are functional genetic variants. ES promotes proteasome-mediated degradation of the transcriptional repressor ID1, increasing expression and release of TSP-1 (thrombospondin 1). ES inhibits PEC migration via an ID1/TSP-1/CD36-dependent pathway, in contrast to proliferation and apoptosis, which require both CD36 and CD47. Collectively, the data implicate ES as a novel negative regulator of ID1 and an upstream propagator of an angiostatic signal cascade converging on CD36 and CD47, providing insight into the cellular and molecular effects of a functional genetic variant linked to altered outcomes in PAH.
Assuntos
Colágeno Tipo VIII/metabolismo , Endostatinas/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Pulmão/metabolismo , Apoptose/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Colágeno Tipo XVIII/metabolismo , Genética Humana/métodos , Humanos , Transdução de Sinais/fisiologiaRESUMO
KEY POINTS: Extracellular matrix is highly remodelled in obesity and associates with the development of metabolic disorders, such as insulin resistance. Previously, we have shown that the lack of specific collagen XVIII isoforms impairs adipocyte differentiation in mice. Here, we show that mice lacking the medium and long isoforms of collagen XVIII develop insulin resistance and glucose intolerance and show elevated serum triglycerides and fat accumulation in the liver. We report that collagen XVIII-deficient mice have increased heat production at low temperatures. These results reveal a new role for collagen XVIII in the regulation of glucose and lipid metabolism, and they expand the understanding of the development of metabolic disorders. ABSTRACT: Liver and adipose tissues play important roles in the regulation of systemic glucose and lipid metabolism. Extracellular matrix synthesis and remodelling are significantly altered in these tissues in obesity and type 2 diabetes. Collagen XVIII is a ubiquitous extracellular matrix component, and it occurs in three isoforms which differ in terms of molecular size, domain structure and tissue distribution. We recently showed that, in mice, the lack of collagen XVIII, and especially its medium and long isoforms, leads to reduced adiposity and dyslipidaemia. To address the metabolic consequences of these intriguing observations, we assessed whole-body glucose homeostasis in mice challenged with a high-fat diet and in normal physiological conditions. We observed that, in the high caloric diet, the overall adiposity was decreased by 30%, serum triglyceride values were threefold higher and the steatotic area in liver was twofold larger in collagen XVIII knockout mice compared with controls. We demonstrated that mice lacking either all three collagen XVIII isoforms, or specifically, the medium and long isoforms develop insulin resistance and glucose intolerance. Furthermore, we found that ablation of collagen XVIII leads to increased heat production in low temperatures and to reduction of the high blood triglyceride levels of the knockout mice to the level of wild-type mice. Our data indicate that collagen XVIII plays a role in the regulation of glucose tolerance, insulin sensitivity and lipid homeostasis, principally through its ability to regulate the expansion of the adipose tissue. These findings advance the understanding of metabolic disorders.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Lipodistrofia , Tecido Adiposo/metabolismo , Animais , Colágeno Tipo XVIII/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica , Glucose/metabolismo , Homeostase , Metabolismo dos Lipídeos , Lipodistrofia/metabolismo , Fígado/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Primary angle-closure glaucoma (PACG) is a common form of glaucoma in the Far East. Its defining feature is iridocorneal angle closure. In addition to PACG, indications of angle closure are included in the diagnostic criteria of related conditions primary angle-closure suspect (PACS) and primary angle closure (PAC). To the best of our knowledge, a causative gene for iridocorneal angle closure in humans has not been identified. This study aimed to identify the genetic cause of iridocorneal angle closure in a pedigree with at least 10 individuals diagnosed with PACS, PAC or PACG. Results of linkage analysis, segregation analysis of 44 novel variations, whole exome sequencing of 10 individuals, screenings of controls and bioinformatics predictions identified a mutation in COL18A1 that encodes collagen type XVIII as the most likely cause of angle closure in the pedigree. The role of COL18A1 in the etiology of Knobloch syndrome (KS) that is consistently accompanied by optic anomalies, available functional data on the encoded protein and the recognized role of collagens and the extracellular matrix in glaucoma pathogenesis supported the proposed role of the COL18A1 mutation in the pedigree. Subsequent identification of other COL18A1 mutations in PACS affected individuals of two unrelated families further supported that COL18A1 may affect angle closure. These PACS individuals were parents and grandparents of KS-affected children. In conclusion, a gene that affects angle closure in humans, a critical feature of PACG, has been identified. The findings also reinforce the importance of collagens in eye features and functions.
Assuntos
Colágeno Tipo VIII/metabolismo , Colágeno Tipo XVIII/metabolismo , Glaucoma de Ângulo Fechado/genética , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Colágeno Tipo VIII/genética , Colágeno Tipo XVIII/genética , Análise Mutacional de DNA , Olho/metabolismo , Feminino , Glaucoma de Ângulo Fechado/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Collagen XVIII (ColXVIII) is a non-fibrillar collagen and proteoglycan that exists in three isoforms: short, medium and long. The medium and long isoforms contain a unique N-terminal domain of unknown function, DUF959, and our sequence-based secondary structure predictions indicated that DUF959 could be an intrinsically disordered domain. Recombinant DUF959 produced in mammalian cells consisted of â¼50% glycans and had a molecular mass of 63â kDa. Circular dichroism spectroscopy confirmed the disordered character of DUF959, and static light scattering indicated a monomeric state for glycosylated DUF959 in solution. Small-angle X-ray scattering showed DUF959 to be a highly extended, flexible molecule with a maximum dimension of â¼23â nm. Glycosidase treatment demonstrated considerable amounts of O-glycosylation, and expression of DUF959 in HEK293 SimpleCells capable of synthesizing only truncated O-glycans confirmed the presence of N-acetylgalactosamine-type O-glycans. The DUF959 sequence is characterized by numerous Ser and Thr residues, and this accounts for the finding that half of the recombinant protein consists of glycans. Thus, the medium and long ColXVIII isoforms contain at their extreme N-terminus a disordered, elongated and highly O-glycosylated mucin-like domain that is not found in other collagens, and we suggest naming it the Mucin-like domain in ColXVIII (MUCL-C18). As intrinsically disordered regions and their post-translational modifications are often involved in protein interactions, our findings may point towards a role of the flexible mucin-like domain of ColXVIII as an interaction hub affecting cell signaling. Moreover, the MUCL-C18 may also serve as a lubricant at cell-extracellular matrix interfaces.
Assuntos
Colágeno Tipo XVIII/química , Colágeno Tipo XVIII/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Colágeno Tipo XVIII/genética , Glicosilação , Células HEK293 , Humanos , Camundongos , Polissacarídeos/química , Polissacarídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios XRESUMO
The basement membrane (BM) is composed of various extracellular molecules and regulates tissue regeneration and maintenance. Here, we demonstrate that collagen XVIII was spatiotemporally expressed in the BM during skin wound healing in a mouse excisional wound-splinting model. Re-epithelialization was detected at days 3 and 6 post-wounding. The ultrastructure of epidermal BM was discontinuous at day 3, whereas on day 6 a continuous BM was observed in the region proximal to the wound edge. Immunohistochemistry demonstrated that collagen XVIII was deposited in the BM zone beneath newly forming epidermis in day 3 and 6 wounds. Laminin-332, known to be the earliest BM component appearing in wounds, was colocalized with collagen XVIII in the epidermal BM zone at days 3 and 6. The deposition of α1(IV) collagen and nidogen-1 in the epidermal BM zone occurred later than that of collagen XVIII. We also observed the short isoform of collagen XVIII in the epidermal BM zone at day 3 post-wounding. Collectively, our results suggested that collagen XVIII plays a role in the formation of the dermal-epidermal junction during re-epithelialization, and that it is the short isoform that is involved in the early phase of re-epithelialization.
Assuntos
Membrana Basal/fisiologia , Colágeno Tipo XVIII/metabolismo , Células Epidérmicas/metabolismo , Cicatrização/fisiologia , Animais , Membrana Basal/ultraestrutura , Epiderme/patologia , Junções Intercelulares/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Epithelial keratinization involves complex cellular modifications that provide protection against pathogens and chemical and mechanical injuries. In the oral cavity, keratinized mucosa is also crucial to maintain healthy periodontal or peri-implant tissues. In this study, we investigated the roles of type XVIII collagen, a collagen-glycosaminoglycan featuring an extracellular matrix component present in the basement membrane, in oral mucosal keratinization. Histological analysis of keratinized and non-keratinized oral mucosa showed that type XVIII collagen was highly expressed in keratinized mucosa. Additionally, a 3D culture system using human squamous carcinoma cells (TR146) was used to evaluate and correlate the changes in the expression of type XVIII collagen gene, COL18A1, and epithelial keratinization-related markers, e.g., keratin 1 (KRT1) and 10 (KRT10). The results showed that the increase in COL18A1 expression followed the increase in KRT1 and KRT10 mRNA levels. Additionally, loss-of-function analyses using silencing RNA targeting COL18A1 mRNA and a Col18-knockout (KO) mouse revealed that the absence of type XVIII collagen induces a dramatic decrease in KRT10 expression as well as in the number and size of keratohyalin granules. Together, the results of this study demonstrate the importance of type XVIII collagen in oral mucosal keratinization.
Assuntos
Colágeno Tipo XVIII/metabolismo , Grânulos Citoplasmáticos/metabolismo , Queratinas/metabolismo , Mucosa Bucal/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Colágeno Tipo XVIII/genética , Imunofluorescência , Humanos , Camundongos , Camundongos KnockoutRESUMO
Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.
Assuntos
Colágeno Tipo XVIII/metabolismo , Endostatinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Colágeno Tipo XVIII/química , Espaço Extracelular/enzimologia , Transferência Ressonante de Energia de Fluorescência , Hemangioendotelioma/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Peptídeos/metabolismo , Subunidades Proteicas/metabolismo , ProteóliseRESUMO
Endostatin (EST), an antiangiogenic factor, is enriched in aging kidneys. EST is also an interactive partner of transglutaminase 2 (TG2), an enzyme that cross-links extracellular matrix proteins. Here we tested whether EST and TG2 play a role in the fibrosis of aging. In wild-type mice, aging kidneys exhibited a 2- to 4-fold increase in TG2 paralleled by increased cross-linked extracellular matrix proteins and fibrosis. Mice transgenic to express EST showed renal fibrosis at a young age. One-month delivery of EST via minipumps to young mice showed increased renal fibrosis that became more robust when superimposed on folic acid-induced nephropathy. Upregulated TG2 and impaired renal function were apparent with EST delivery combined with folic acid-induced nephropathy. Subcapsular injection of TG2 and/or EST into kidneys of young mice not only induced interstitial fibrosis, but also increased the proportion of senescent cells. Thus, kidney fibrosis in aging may represent a natural outcome of upregulated EST and TG2, but more likely it appears to be a result of cumulative stresses occurring on the background of synergistically acting geronic (aging) proteins, EST and TG2.
Assuntos
Envelhecimento/metabolismo , Colágeno Tipo XVIII/metabolismo , Endostatinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Nefropatias/patologia , Rim/patologia , Transglutaminases/metabolismo , Animais , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Colágeno Tipo XVIII/genética , Colágeno Tipo XVIII/farmacologia , Endostatinas/genética , Endostatinas/farmacologia , Células Endoteliais , Proteínas da Matriz Extracelular , Fibrose , Ácido Fólico/toxicidade , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genética , Transglutaminases/farmacologia , Regulação para CimaRESUMO
As the second most common skin malignancy, cutaneous squamous cell carcinoma (cSCC) is an increasing health concern, while its pathogenesis at molecular level remains largely unknown. We studied the expression and localisation of two homologous basement membrane (BM) collagens, types XV and XVIII, at different stages of cSCC. These collagens are involved in angiogenesis and tumorigenesis, but their role in cancer development is incompletely understood. Quantitative RT-PCR analysis revealed upregulation of collagen XVIII, but not collagen XV, in primary cSCC cells in comparison with normal human epidermal keratinocytes. In addition, the Ha-ras-transformed invasive cell line II-4 expressed high levels of collagen XVIII mRNA, indicating upregulation in the course of malignant transformation. Immunohistochemical analyses of a large human tissue microarray material showed that collagen XVIII is expressed by tumor cells from grade 1 onwards, while keratinocytes in normal skin and in premalignant lesions showed negative staining for it. Collagen XV appeared instead as deposits in the tumor stroma. Our findings in human cSCCs and in mouse cSCCs from the DMBA-TPA skin carcinogenesis model showed that collagen XVIII, but not collagen XV or the BM markers collagen IV or laminin, was selectively reduced in the tumor vasculature, and this decrease associated significantly with cancer progression. Our results demonstrate that collagens XV and XVIII are expressed in different sites of cSCC and may contribute in a distinct manner to processes related to cSCC tumorigenesis, identifying these collagens as potential biomarkers in the disease.
Assuntos
Membrana Basal/metabolismo , Carcinoma de Células Escamosas/metabolismo , Colágeno Tipo XVIII/metabolismo , Colágeno/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Humanos , CamundongosRESUMO
BACKGROUND: Endostatin, a fragment of collagen XVIII, is an endogenous angiogenesis inhibitor with anti-tumour functions. However, elevated circulating endostatin concentrations have been found in several human cancers including colorectal cancer (CRC). METHODS: Serum endostatin levels were measured by enzyme-linked immunoassay from a series of 143 patients with CRC and from 84 controls, and correlated with detailed clinicopathological features of CRC, serum leukocyte differential count and C-reactive protein (CRP) levels. RESULTS: Patients with CRC had higher serum endostatin levels than the controls (P=0.005), and high levels associated with age, tumour invasion through the muscularis propria and poor differentiation, but not with metastases. Endostatin levels showed a positive correlation with the markers of systemic inflammatory response and a negative correlation with the densities of tumour-infiltrating mast cells and dendritic cells. Collagen XVIII was expressed in tumour stroma most strikingly in blood vessels and capillaries, and in the muscle layer of the bowel wall. CONCLUSIONS: Elevated endostatin levels in CRC correlate with systemic inflammation and invasion through the muscularis propria. Increased endostatin level may be a result of invasion-related cleavage of collagen XVIII expressed in the bowel wall. The negative correlations between serum endostatin and intratumoural mast cells and immature dendritic cells may reflect angiogenesis inhibition by endostatin.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Endostatinas/sangue , Inflamação/sangue , Invasividade Neoplásica , Idoso , Colágeno Tipo XVIII/metabolismo , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Feminino , Humanos , Inflamação/complicações , Masculino , Pessoa de Meia-IdadeRESUMO
Collagen type XVIII (COL18) is an abundant heparan sulfate proteoglycan in vascular basement membranes. Here, we asked (i) if the loss of COL18 would result in blood-brain barrier (BBB) breakdown, pathological alterations of small arteries and capillaries and neuroinflammation as found in cerebral small vessel disease (CSVD) and (ii) if such changes may be associated with remodeling of synapses and neural extracellular matrix (ECM). We found that 5-month-old Col18a1-/- mice had elevated BBB permeability for mouse IgG in the deep gray matter, and intravascular erythrocyte accumulations were observed brain-wide in capillaries and arterioles. BBB permeability increased with age and affected cortical regions and the hippocampus in 12-month-old Col18a1-/- mice. None of the Col18a1-/- mice displayed hallmarks of advanced CSVD, such as hemorrhages, and did not show perivascular space enlargement. Col18a1 deficiency-induced BBB leakage was accompanied by activation of microglia and astrocytes, a loss of aggrecan in the ECM of perineuronal nets associated with fast-spiking inhibitory interneurons and accumulation of the perisynaptic ECM proteoglycan brevican and the microglial complement protein C1q at excitatory synapses. As the pathway underlying these regulations, we found increased signaling through the TGF-ß1/Smad3/TIMP-3 cascade. We verified the pivotal role of COL18 for small vessel wall structure in CSVD by demonstrating the protein's involvement in vascular remodeling in autopsy brains from patients with cerebral hypertensive arteriopathy. Our study highlights an association between the alterations of perivascular ECM, extracellular proteolysis, and perineuronal/perisynaptic ECM, as a possible substrate of synaptic and cognitive alterations in CSVD.
Assuntos
Doenças de Pequenos Vasos Cerebrais , Colágeno Tipo XVIII , Doenças Neuroinflamatórias , Animais , Humanos , Lactente , Camundongos , Doenças de Pequenos Vasos Cerebrais/genética , Doenças de Pequenos Vasos Cerebrais/metabolismo , Colágeno Tipo XVIII/genética , Colágeno Tipo XVIII/metabolismo , Endostatinas , Matriz Extracelular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Camundongos KnockoutRESUMO
Necrotizing enterocolitis (NEC) is typified by mucosal destruction, which subsequently can lead to intestinal necrosis. Prematurity, enteral feeding, and bacterial colonization are the main risk factors and, combined with other stressors, can cause increased intestinal permeability, injury, and an exaggerated inflammatory response. Heme oxygenase-1 (HO-1) mediates intestinal protection due to anti-inflammatory, antioxidative, and antiapoptotic effects of its products carbon monoxide, biliverdin, and bilirubin. This study investigates a possible role of HO-1 in the pathogenesis of NEC using a newborn mouse model. We induced NEC-like intestinal injury in 7-day-old HO-1 heterozygous (HO-1 Het, Hmox1(+/-)) and wild-type (Wt, Hmox1(+/+)) mice by gavage feeding and hypoxic exposures. Control (Con) pups of both genotypes were dam-fed. Intestines of HO-1 Het Con pups appeared predisposed to injury, with higher histological damage scores, more TUNEL-positive cells, and a significant reduction in muscularis externa thickness compared with Wt Con pups. The increase in HO activity after HO-1 induction by the substrate heme or by hypoxic stress was significantly impaired in HO-1 Het pups. After induction of intestinal injury, HO-1 Het pups displayed significantly higher NEC incidence (78 vs. 43%), mortality (83 vs. 54%), and median scores (2.5 vs. 1.5) than Wt NEC pups. PCR array analyses revealed increased expressions of IL-1ß, P-selectin, matrix metallopeptidase 2, collagen type XVIII-α1, serpine 1, and others in NEC-induced HO-1 Het ileal and jejunal tissues. We conclude that a partial HO-1 deficiency promotes experimental NEC-like intestinal injury, possibly mediated by exaggerated inflammation and disruption in tissue repair.
Assuntos
Enterocolite Necrosante/genética , Heme Oxigenase-1/genética , Proteínas de Membrana/genética , Animais , Animais Recém-Nascidos , Apoptose , Colágeno Tipo XVIII/genética , Colágeno Tipo XVIII/metabolismo , Modelos Animais de Doenças , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Genótipo , Heme/metabolismo , Heme Oxigenase-1/deficiência , Heme Oxigenase-1/metabolismo , Hipóxia , Íleo/metabolismo , Íleo/patologia , Escala de Gravidade do Ferimento , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Jejuno/metabolismo , Jejuno/patologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mucosa/patologia , Selectina-P/genética , Selectina-P/metabolismo , Transcrição GênicaRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.
Assuntos
Colágeno Tipo XVIII/metabolismo , Dobramento de Proteína , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Cromatografia em Gel , Colágeno Tipo XVIII/genética , Colágeno Tipo XVIII/isolamento & purificação , Colágeno Tipo XVIII/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Escherichia coli/genética , Expressão Gênica , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
BACKGROUND: The objective of this study was to assess the possibility of predicting histological characteristics of meningiomas on the basis of preoperative MRI and the correlation of the expression of vascular endothelial growth factor (VEGF) and collagen XVIII with histological parameters already established as predictive of the course of these tumors. METHODS: Expression of VEGF and collagen XVIII as well as other histological characteristics was examined in meningioma tissues from 20 patients. Preoperative MRI, including dynamic imaging of contrast enhancement, was analyzed. Times to maximum enhancement and maximum intensity increase were noted from dynamic imaging. The relative intensity of the tumor in fluid-attenuated inversion recovery (FLAIR), T2-weighted and contrast enhanced T1-weighted images, as well as volumes of tumor and edema, was calculated. The edema-tumor volume ratio was defined as the edema index (EI). RESULTS: Both VEGF and collagen XVIII were expressed in all meningioma samples. Edema was present in 60 % of cases. The strongest correlation of VEGF expression was to EI. Among histological parameters, microvessel density (MVD) and cellularity correlated moderately with VEGF. Collagen XVIII expression correlated strongly with the maximal intensity increase after contrast agent administration (ρ = 0.71, P = 0.001) as well as with MVD and intensity of the meningioma on FLAIR images. CONCLUSION: Meningiomas with faster and more intense enhancement in dynamic studies, indicative of good tumor blood supply and permeability of vasculature, are associated with high levels of collagen XVIII and VEGF expression. Occurrence of peritumoral edema in meningiomas is strongly correlated with expression of VEGF.
Assuntos
Edema Encefálico/patologia , Colágeno Tipo XVIII/metabolismo , Meningioma/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Edema Encefálico/etiologia , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Meningioma/irrigação sanguínea , Meningioma/patologia , Meningioma/cirurgia , Pessoa de Meia-IdadeRESUMO
Collagen XVIII (ColXVIII) is a component of the extracellular matrix implicated in embryogenesis and control of tissue homoeostasis. We now provide evidence that ColXVIII has a specific role in renal branching morphogenesis as observed in analyses of total and isoform-specific knockout embryos and mice. The expression of the short and the two longer isoforms differ temporally and spatially during renal development. The lack of ColXVIII or its specific isoforms lead to congenital defects in the 3D patterning of the ureteric tree where the short isoform plays a prominent role. Moreover, the ex vivo data suggests that ColXVIII is involved in the kidney epithelial tree patterning via its N-terminal domains, and especially the Thrombospondin-1-like domain common to all isoforms. This morphogenetic function likely involves integrins expressed in the ureteric epithelium. Altogether, the results point to an important role for ColXVIII in the matrix-integrin-mediated functions regulating renal development.
Assuntos
Colágeno Tipo XVIII , Rim , Isoformas de Proteínas , Animais , Camundongos , Colágeno Tipo XVIII/genética , Colágeno Tipo XVIII/metabolismo , Integrinas , Rim/embriologia , Rim/metabolismo , Morfogênese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ureter/embriologia , Ureter/metabolismoRESUMO
The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII was markedly upregulated in human breast cancer (BC) and was closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of epidermal growth factor receptor tyrosine kinase (ErbB) signaling and show that it forms a complex with ErbB1 and -2 (also known as EGFR and human epidermal growth factor receptor 2 [HER2]) and α6-integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies using Col18a1 mouse models crossed with the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) mammary carcinogenesis model showed that ColXVIII promoted BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.
Assuntos
Neoplasias da Mama , Colágeno Tipo XVIII , Camundongos , Animais , Humanos , Feminino , Colágeno Tipo XVIII/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/metabolismo , Transformação Celular Neoplásica , Transdução de SinaisRESUMO
Rac1 activity, polarity, lamellipodial dynamics, and directed motility are defective in keratinocytes exhibiting deficiency in ß4 integrin or knockdown of the plakin protein Bullous Pemphigoid Antigen 1e (BPAG1e). The activity of Rac, formation of stable lamellipodia, and directed migration are restored in ß4 integrin-deficient cells by inducing expression of a truncated form of ß4 integrin, which lacks binding sites for BPAG1e and plectin. In these same cells, BPAG1e, the truncated ß4 integrin, and type XVII collagen (Col XVII), a transmembrane BPAG1e-binding protein, but not plectin, colocalize along the substratum-attached surface. This finding suggested to us that Col XVII mediates the association of BPAG1e and α6ß4 integrin containing the truncated ß4 subunit and supports directed migration. To test these possibilities, we knocked down Col XVII expression in keratinocytes expressing both full-length and truncated ß4 integrin proteins. Col XVII-knockdown keratinocytes exhibit a loss in BPAG1e-α6ß4 integrin interaction, a reduction in lamellipodial stability, an impairment in directional motility, and a decrease in Rac1 activity. These defects are rescued by a mutant Col XVII protein truncated at its carboxyl terminus. In summary, our results suggest that in motile cells Col XVII recruits BPAG1e to α6ß4 integrin and is necessary for activation of signaling pathways, motile behavior, and lamellipodial stability.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular/fisiologia , Colágeno Tipo XVIII/metabolismo , Proteínas do Citoesqueleto/metabolismo , Integrina alfa6beta4/metabolismo , Queratinócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Colágeno Tipo XVIII/genética , Proteínas do Citoesqueleto/genética , Distonina , Técnicas de Silenciamento de Genes , Humanos , Integrina alfa6beta4/genética , Queratinócitos/citologia , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína , Pseudópodes/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
In diabetes the endothelium is either chronically or transiently exposed to hyperglycemic conditions. In addition, endothelial dysfunction in diabetes is related to changes in the inflammatory response and the turnover of extracellular matrix. This study was undertaken to study the effects of inflammatory stimuli on one particular matrix component, the heparan sulfate (HS) proteoglycans (PGs) synthesized by primary human umbilical cord vein endothelial cells (HUVEC). Such cells were cultured in vitro in 5 mM and 25 mM glucose. The latter concentration was used to mimic hyperglycemic conditions in short-term experiments. HUVEC were also cultured in the presence of the inflammatory agents tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 1ß (IL-1ß) and transforming growth factor ß (TGF-ß). The cells were labeled with (35)S-sulfate and (35)S-PGs were recovered for further analyses. The major part of the (35)S-PGs was secreted to the medium, irrespective of type of stimuli. Secreted (35)S-PGs were therefore isolated and subjected to further analyses. TNF-α and IL-1α slightly increased the release of (35)S-PGs to the culture medium, whereas IL-1ß treatment gave a significant increase. The different treatments neither changed the ratio of (35)S-HS and (35)S-chondroitin sulfate (CS) nor the macromolecular properties of the (35)S-PGs. However, the (35)S-HS chains were slightly increased in size after TNF-α treatment, and slightly decreased after TGF-ß treatment, but not affected by the other treatments. Compositional analysis of labeled disaccharides showed changes in the amount of 6-O-sulfated glucosamine residues after treatment with TNF-α, IL-1α and IL-1ß. Western immunoblotting showed that major HSPGs recovered from these cells were collagen XVIII, perlecan and agrin, and that secretion of these distinct PGs was increased after IL-1ß stimulation. Hence, short term inflammatory stimuli increased the release of HSPGs in HUVEC and affected both the size and sulfation pattern of HS, depending on type of stimuli.