RESUMO
Type 1 Diabetes (T1D) is an autoimmune disease marked by direct elimination of insulin-producing ß cells by autoreactive T effectors. Recent T1D clinical trials utilizing autologous Tregs transfers to restore immune balance and improve disease has prompted us to design a novel Tregs-based antigen-specific T1D immunotherapy. We engineered a Chimeric Antigen Receptor (CAR) expressing a single-chain Fv recognizing the human pancreatic endocrine marker, HPi2. Human T cells, transduced with the resultant HPi2-CAR, proliferated and amplified Granzyme B accumulation when co-cultured with human, but not mouse ß cells. Furthermore, following exposure of HPi2-CAR transduced cells to islets, CD8+ lymphocytes demonstrated enhanced CD107a (LAMP-1) expression, while CD4+ cells produced increased levels of IL-2. HPi2-CAR Tregs failed to maintain expansion due to a persistent tonic signaling from the CAR engagement to unexpectantly HPi2 antigen present on Tregs. Overall, we show lack of functionality of HPi2-CAR and highlight the importance of careful selection of CAR recognition driver for the sustainable activity and expandability of engineered T cells.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Protaminas/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Humanos , Tolerância Imunológica/imunologia , Imunoterapia Adotiva/métodos , Ilhotas Pancreáticas , Pâncreas/citologia , Pâncreas/metabolismo , Protaminas/metabolismo , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
Type 1 diabetes (T1D) is characterized by massive destruction of insulin-producing ß cells by autoreactive T lymphocytes, arising via defective immune tolerance. Therefore, effective anti-T1D therapeutics should combine autoimmunity-preventing and insulin production-restoring properties. We constructed a cell-permeable PDX1-FOXP3-TAT fusion protein (FP) composed of two transcription factors: forkhead box P3 (FOXP3), the master regulator of differentiation and functioning of self-tolerance-promoting Tregs, and pancreatic duodenal homeobox-1 (PDX1), the crucial factor supporting ß cell development and maintenance. The FP was tested in vitro and in a non-obese diabetic mouse T1D model. In vitro, FP converted naive CD4+ T cells into a functional "Treg-like" subset, which suppressed cytokine secretion, downregulated antigen-specific responses, and curbed viability of diabetogenic effector cells. In hepatic stem-like cells, FP potentiated endocrine transdifferentiation, inducing expression of Insulin2 and other ß lineage-specific genes. In vivo, FP administration to chronically diabetic mice triggered (1) a significant elevation of insulin and C-peptide levels, (2) the formation of insulin-containing cell clusters in livers, and (3) a systemic anti-inflammatory shift (higher Foxp3+CD4+CD25+ T cell frequencies, elevated rates of IL-10-producing cells, and reduced rates of IFN-γ-secreting cells). Overall, in accordance with its design, PDX1-FOXP3-TAT FP delivered both Treg-stabilizing anti-autoimmune and de novo insulin-producing effects, proving its anti-T1D therapeutic potential.
Assuntos
Autoimunidade , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Homeodomínio/metabolismo , Secreção de Insulina , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transativadores/metabolismo , Animais , Microambiente Celular/imunologia , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/genética , Hepatócitos/metabolismo , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fenótipo , Ligação Proteica , Proteínas Recombinantes de Fusão , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transativadores/genéticaRESUMO
Ag-specific activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the costimulation step, have been associated with many autoimmune conditions, including type 1 diabetes (T1D). Recently, we demonstrated that the phenotype of impaired negative costimulation, due to reduced levels of V-set domain-containing T cell activation inhibitor 1 (VTCN1) protein on APCs, is shared between diabetes-susceptible NOD mice and human T1D patients. In this study, we show that a similar process takes place in the target organ, as both α and ß cells within pancreatic islets gradually lose their VTCN1 protein during autoimmune diabetes development despite upregulation of the VTCN1 gene. Diminishment of functional islet cells' VTCN1 is caused by the active proteolysis by metalloproteinase N-arginine dibasic convertase 1 (NRD1) and leads to the significant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, alternatively, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 negative costimulation, which affects both lymphoid and peripheral target tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to upregulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/imunologia , Animais , Apresentação de Antígeno , Citocinas/biossíntese , Diabetes Mellitus Tipo 1/fisiopatologia , Células Secretoras de Glucagon/imunologia , Humanos , Células Secretoras de Insulina/imunologia , Ilhotas Pancreáticas/metabolismo , Ativação Linfocitária , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos Endogâmicos NOD , Proteólise , Linfócitos T/imunologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/metabolismoRESUMO
Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of ß-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and ß-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.
Assuntos
Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Diferenciação Celular/fisiologia , Humanos , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologiaRESUMO
Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin alphaB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS.
Assuntos
Células Apresentadoras de Antígenos/patologia , Encéfalo/imunologia , Inflamação/enzimologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Esclerose Múltipla/etiologia , Pró-Proteína Convertases/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Proteínas Ligadas por GPI , Humanos , Inflamação/etiologia , Metaloproteinases da Matriz Associadas à Membrana/genética , Microdomínios da Membrana , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Proteína Básica da Mielina/metabolismo , Transdução de Sinais , Distribuição Tecidual , Regulação para CimaRESUMO
Activated insulin-specific CD8(+) T cells (IS-CD8(+) cells) home to the pancreas, destroy beta cells, and cause rapid diabetes upon transfer into diabetes-prone NOD mice. Surprisingly, they also cause diabetes in mouse strains that are free of preexistent inflammation. Thus, we hypothesized that islet-specific homing may be in part dependent on IS-CD8(+) cells' recognition of the cognate major histocompatibility complex (MHC)/peptide complexes presented by pancreatic endothelial cells, which acquire the antigen (insulin) from beta cells. In fact, islet-specific homing was abrogated in mice that lack MHC class I expression, or presentation of the specific peptide, or have impaired insulin secretion. Moreover, we found that IS-CD8(+) cells directly recognized pancreatic endothelial cells in islet organ cultures. Triggering of IS-CD8(+) cells' T cell receptor (TCR) led to activation of integrins expressed by these cells. In addition, chemokines, particularly SLC (CCL21), were also required for IS-CD8(+) cells' adhesion to endothelial monolayers and for successful homing in vivo. Thus, signaling through TCR and chemokine receptors work in concert to assure firm adhesion of T cells to the pancreatic endothelium. The antigen cross-presentation ability of endothelia may therefore contribute to the specificity of homing of activated T lymphocytes to the tissues where antigens are generated by other cell types.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Quimiotaxia , Endotélio Vascular/imunologia , Insulina/imunologia , Ilhotas Pancreáticas/imunologia , Animais , Antígenos CD , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Adesão Celular/fisiologia , Células Cultivadas , Técnicas de Cultura , Diabetes Mellitus Experimental/imunologia , Endoglina , Endotélio Vascular/citologia , Feminino , Genes MHC Classe I , Transportador de Glucose Tipo 2 , Ilhotas Pancreáticas/irrigação sanguínea , Complexo Principal de Histocompatibilidade , Masculino , Camundongos , Camundongos Endogâmicos , Proteínas de Transporte de Monossacarídeos/metabolismo , Pâncreas/irrigação sanguínea , Pâncreas/citologia , Pâncreas/imunologia , Pâncreas/metabolismo , Pâncreas/patologia , Peptídeos/metabolismo , Toxina Pertussis/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Superfície Celular , Receptores de Quimiocinas/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Neoplasms have developed strategies to protect themselves against the complement-mediated host immunity. Invasion- and metastasis-promoting membrane type-1 (MT1) matrix metalloproteinase (MMP) is strongly associated with many metastatic cancer types. The relative importance of the individual functions of MT1-MMP in metastasis was, however, unknown. We have now determined that the expression of murine MT1-MMP in murine melanoma B16F1 cells strongly increased the number of metastatic loci in the lungs of syngeneic C57BL/6 mice. In contrast, MT1-MMP did not affect the number of metastatic loci in complement-deficient C57BL/6-C3-/- mice. Our results indicated, for the first time, that the anticomplement activity of MT1-MMP played a significant role in promoting metastasis in vivo and determined the relative importance of the anticomplement activity in the total metastatic effect of this multifunctional proteolytic enzyme. We believe that our results shed additional light on the functions of MT1-MMP in cancer and clearly make this protease a promising drug target in metastatic malignancies.
Assuntos
Complemento C3/imunologia , Proteínas Inativadoras do Complemento/imunologia , Metaloproteinases da Matriz/imunologia , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/imunologia , Animais , Complemento C3/deficiência , Complemento C3/genética , Proteínas Inativadoras do Complemento/genética , Proteínas Inativadoras do Complemento/metabolismo , Fibrossarcoma/enzimologia , Fibrossarcoma/genética , Fibrossarcoma/imunologia , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Metaloproteinase 14 da Matriz , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , TransfecçãoRESUMO
Estrogens have many cellular functions, including their interactions with estrogen receptors alpha and beta (ERalpha and ERbeta). Earlier, we determined that the estrogen-ER complex stimulates the transcriptional activity of the matrix metalloproteinase 26 (MMP-26) gene promoter. We then determined that ERbeta is susceptible to MMP-26 proteolysis whereas ERalpha is resistant to the protease. MMP-26 targets the NH(2)-terminal region of ERbeta coding for the divergent NH(2)-terminal A/B domain that is responsible for the ligand-independent transactivation function. As a result, MMP-26 proteolysis generates the COOH-terminal fragments of ERbeta. Immunohistochemical analysis of tissue microarrays derived from 121 cancer patients corroborated these data and revealed an inverse correlation between the ERalpha-dependent expression of MMP-26 and the levels of the intact ERbeta in breast carcinomas. MMP-26 is not expressed in normal mammary epithelium. The levels of MMP-26 are strongly up-regulated in ductal carcinoma in situ (DCIS). In the course of further disease progression through stages I to III, the expression of MMP-26 decreases. In contrast to many tumor-promoting MMPs, the expression of MMP-26 in DCIS correlated with a longer patient survival. Our data suggest the existence of an MMP-26-mediated intracellular pathway that targets ERbeta and that MMP-26, a novel and valuable cancer marker, contributes favorably to the survival of the ERalpha/beta-positive cohort of breast cancer patients.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma Ductal de Mama/metabolismo , Receptor beta de Estrogênio/metabolismo , Metaloproteinases da Matriz/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Carcinoma in Situ/enzimologia , Carcinoma in Situ/patologia , Carcinoma Ductal de Mama/enzimologia , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Humanos , Metaloproteinases da Matriz Secretadas , Estadiamento de Neoplasias , Taxa de SobrevidaRESUMO
It has been well established that invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP), a multifunctional membrane-tethered enzyme, functions in cancer cells as a mediator of pericellular proteolysis and directly cleaves several cell surface receptors, including CD44. In this report, we confirm that adhesion of diabetogenic T cells promotes the activation of endogenous MT1-MMP. Activated protease then cleaves CD44 in adherent T cells. We have validated that the T cell CD44 receptor is critical for the adhesion of diabetogenic insulin-specific, CD8-positive, K(d)-restricted cells to the matrix as well as for the subsequent transmigration of the adherent T cells through the endothelium and homing of the transmigrated T cells into the pancreatic islets. We have determined that the inhibition of MT1-MMP by low dosages of AG3340 (a subnanomolar range hydroxamate inhibitor of MMPs that has been widely tested in cancer patients) inhibited both T cell MT1-MMP activity and MT1-MMP-dependent shedding of CD44, immobilized T cells on the endothelium, repressed the homing of diabetogenic T cells into the pancreatic islets, reduced insulitis and mononuclear cell infiltration, and promoted either the recovery or the rejuvenation of the functional insulin-producing beta cells in diabetic NOD mice with freshly developed type I diabetes (IDDM). We believe our data constitute a mechanistic and substantive rationale for clinical trials of selected MT1-MMP inhibitors in the therapy of IDDM in humans.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Animais , Adesão Celular , Sobrevivência Celular , Ativação Enzimática , Citometria de Fluxo , Receptores de Hialuronatos/fisiologia , Camundongos , Camundongos Endogâmicos NODRESUMO
OBJECTIVE: Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium. APPROACH AND RESULTS: A vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (whc allele) utilizing a Tie2-cre recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0µm2 alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330µm2 oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg-1*d-1, for 5 weeks) reduced coronary calcium (78838 vs.144622µm2) and lipids (30754 vs. 77317µm2); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls. CONCLUSIONS: Endogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.
Assuntos
Fosfatase Alcalina/metabolismo , Doença da Artéria Coronariana/etiologia , Hiperlipoproteinemia Tipo II/patologia , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/genética , Animais , Análise Química do Sangue , Peso Corporal/efeitos dos fármacos , Calcificação Fisiológica , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Citocinas/sangue , Dieta Aterogênica , Modelos Animais de Doenças , Ecocardiografia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Inibidores Enzimáticos/farmacologia , Humanos , Hiperlipoproteinemia Tipo II/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/enzimologia , Miocárdio/metabolismo , Miocárdio/patologia , Efeito Placebo , Receptores de LDL/genética , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Proteases exert control over cell behavior and affect many biological processes by making proteolytic modification of regulatory proteins. The purpose of this paper is to describe novel, important functions of matrix metalloproteinase (MMP)-26. alpha1-Antitrypsin (AAT) is a serpin, the primary function of which is to regulate the activity of neutrophil/leukocyte elastase. Insufficient antiprotease activity because of AAT deficiency in the lungs is a contributing factor to early-onset emphysema. We recently discovered that AAT is efficiently cleaved by a novel metalloproteinase, MMP-26, which exhibits an unconventional PH(81)CGVPD Cys switch motif and is autocatalytically activated in cells and tissues. An elevated expression of MMP-26 in macrophages and polymorphonuclear leukocytes supports the functional role of MMP-26 in the AAT cleavage and inflammation. We have demonstrated a direct functional link of MMP-26 expression with an estrogen dependency and confirmed the presence of the estrogen-response element in the MMP-26 promoter. Immunostaining of tumor cell lines and biopsy specimen microarrays confirmed the existence of the inverse correlations of MMP-26 and AAT in cells/tissues. An expression of MMP-26 in the estrogen-dependent neoplasms is likely to contribute to the inactivation of AAT, to the follow-up liberation of the Ser protease activity, and because of these biochemical events, to promote matrix destruction and malignant progression. In summary, we hypothesize that MMP-26, by cleaving and inactivating the AAT serpin, operates as a unique functional link that regulates a coordinated interplay between Ser and metalloproteinases in estrogen-dependent neoplasms.
Assuntos
Estrogênios/fisiologia , Metaloproteinases da Matriz/fisiologia , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias/enzimologia , alfa 1-Antitripsina/metabolismo , Linhagem Celular Tumoral , Humanos , Macrófagos/enzimologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/isolamento & purificação , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Secretadas , Modelos Moleculares , Neoplasias/genética , Neutrófilos/enzimologia , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Ectopic vascular calcification is a common condition associated with aging, atherosclerosis, diabetes, and/or chronic kidney disease. Smooth muscle cells are the best characterized source of osteogenic progenitors in the vasculature; however, recent studies suggest that cells of endothelial origin can also promote calcification. To test this, we sought to increase the osteogenic potential of endothelial cells by overexpressing tissue-nonspecific alkaline phosphatase (TNAP), a key enzyme that regulates biomineralization, and to determine the pathophysiological effect of endothelial TNAP on vascular calcification and cardiovascular function. METHODS AND RESULTS: We demonstrated previously that mice transgenic for ALPL (gene encoding human TNAP) develop severe arterial medial calcification and reduced viability when TNAP is overexpressed in smooth muscle cells. In this study, we expressed the ALPL transgene in endothelial cells following endothelial-specific Tie2-Cre recombination. Mice with endothelial TNAP overexpression survived well into adulthood and displayed generalized arterial calcification. Genes associated with osteochondrogenesis (Runx2, Bglap, Spp1, Opg, and Col2a1) were upregulated in the aortas of endothelial TNAP animals compared with controls. Lesions in coronary arteries of endothelial TNAP mice showed immunoreactivity to Runx2, osteocalcin, osteopontin, and collagen II as well as increased deposition of sialoproteins revealed by lectin staining. By 23 weeks of age, endothelial TNAP mice developed elevated blood pressure and compensatory left ventricular hypertrophy with preserved ejection fraction. CONCLUSIONS: This study presented a novel genetic model demonstrating the osteogenic potential of TNAP-positive endothelial cells in promoting pathophysiological vascular calcification.
Assuntos
Fosfatase Alcalina/metabolismo , Calcinose/metabolismo , Endotélio Vascular/metabolismo , Doença Arterial Periférica/metabolismo , Animais , Calcinose/etiologia , Calcinose/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Doença Arterial Periférica/etiologia , Doença Arterial Periférica/patologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
T-cell responses directed against insulin-secreting pancreatic ß-cells are the key events highlighting type 1 diabetes (T1D). Therefore, a defective control of T-cell activation is thought to underlie T1D development. Recent studies implicated a B7-like negative costimulatory protein, V-set domain-containing T-cell activation inhibitor-1 (VTCN1), as a molecule capable of inhibiting T-cell activation and, potentially, an important constituent in experimental models of T1D. Here, we unravel a general deficiency within the VTCN1 pathway that is shared between diabetes-prone mice and a subset of T1D patients. Gradual loss of membrane-tethered VTCN1 from antigen-presenting cells combined with an increased release of soluble VTCN1 (sVTCN1) occurs in parallel to natural T1D development, potentiating hyperproliferation of diabetogenic T cells. Mechanistically, we demonstrate that the loss of membrane-tethered VTCN1 is linked to proteolytic cleavage mediated by the metalloproteinase nardilysin. The cleaved sVTCN1 fragment was detected at high levels in the peripheral blood of 53% T1D patients compared with only 9% of the healthy subjects. Elevated blood sVTCN1 levels appeared early in the disease progression and correlated with the aggressive pace of disease, highlighting the potential use of sVTCN1 as a new T1D biomarker, and identifying nardilysin as a potential therapeutic target.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Metaloendopeptidases/metabolismo , Transdução de Sinais/fisiologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/metabolismo , Adolescente , Adulto , Animais , Biomarcadores/metabolismo , Criança , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T/metabolismo , Adulto JovemRESUMO
The kallikrein-kinin system (KKS) comprises a cascade of proteolytic enzymes and biogenic peptides that regulate several physiological processes. Over-expression of tissue kallikrein-1 and modulation of the KKS shows beneficial effects on insulin sensitivity and other parameters relevant to type 2 diabetes mellitus. However, much less is known about the role of kallikreins, in particular tissue kallikrein-1, in type 1 diabetes mellitus (T1D). We report that chronic administration of recombinant human tissue kallikrein-1 protein (DM199) to non-obese diabetic mice delayed the onset of T1D, attenuated the degree of insulitis, and improved pancreatic beta cell mass in a dose- and treatment frequency-dependent manner. Suppression of the autoimmune reaction against pancreatic beta cells was evidenced by a reduction in the relative numbers of infiltrating cytotoxic lymphocytes and an increase in the relative numbers of regulatory T cells in the pancreas and pancreatic lymph nodes. These effects may be due in part to a DM199 treatment-dependent increase in active TGF-beta1. Treatment with DM199 also resulted in elevated C-peptide levels, elevated glucagon like peptide-1 levels and a reduction in dipeptidyl peptidase-4 activity. Overall, the data suggest that DM199 may have a beneficial effect on T1D by attenuating the autoimmune reaction and improving beta cell health.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/imunologia , Imunomodulação/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Calicreínas Teciduais/farmacologia , Animais , Autoimunidade/efeitos dos fármacos , Biomarcadores , Glicemia/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diabetes Mellitus Tipo 1/tratamento farmacológico , Feminino , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Proteínas Recombinantes/administração & dosagem , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Calicreínas Teciduais/administração & dosagem , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Modulation of the kallikrein-kinin system (KKS) has been shown to have beneficial effects on glucose homeostasis and several other physiological responses relevant to the progression of type 2 diabetes mellitus (T2D). The importance of bradykinin and its receptors in mediating these responses is well documented, but the role of tissue kallikrein-1, the protease that generates bradykinin in situ, is much less understood. We developed and tested DM199, recombinant human tissue kallikrein-1 protein (rhKLK-1), as a potential novel therapeutic for T2D. Hyperinsulinemic-euglycemic clamp studies suggest that DM199 increases whole body glucose disposal in non-diabetic rats. Single-dose administration of DM199 in obese db/db mice and ZDF rats, showed an acute, dose-dependent improvement in whole-body glucose utilization. Sub-acute dosing for a week in ZDF rats improved glucose utilization, with a concomitant rise in fasting insulin levels and HOMA1-%B scores. After cessation of sub-acute dosing, fasting blood glucose levels were significantly lower in ZDF rats during a drug wash-out period. Our studies show for the first time that DM199 administration results in acute anti-hyperglycemic effects in several preclinical models, and demonstrate the potential for further development of DM199 as a novel therapeutic for T2D.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacocinética , Calicreínas Teciduais/farmacologia , Animais , Glicemia , Células CHO , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Camundongos , Camundongos Obesos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
This study tested the hypothesis that membrane-tethered type-1 matrix metalloproteinase (MT1-MMP)-induced proteolysis of T cell CD44 is important for defining the migration and function of autoreactive T cells, including diabetogenic, insulin-specific and K(d)-restricted IS-CD8(+) cells. To confirm the importance of MT1-MMP proteolysis of CD44 in type 1 diabetes (T1D), the anti-diabetic effects of three MMP inhibitors (3(S)-2,2-dimethyl-4[4-pyridin-4-yloxy-benzenesulfonyl]-thiomorpholine-3-carboxylic acid hydroxamate [AG3340], 2-(4-phenoxyphenylsulfonylmethyl) thiirane [SB-3CT] and epigallocatechin-3-gallate [EGCG]) were compared using an adoptive diabetes transfer model in non-obese diabetic (NOD) mice. Only AG3340 was capable of inhibiting both the activity of MT1-MMP and the shedding of CD44 in T cells; and the transendothelial migration and homing of IS-CD8(+) T cells into the pancreatic islets. SB-3CT and EGCG were incapable of inhibiting T cell MT1-MMP efficiently. As a result, AG3340 alone, but not SB-3CT or EGCG, delayed the onset of transferred diabetes in NOD mice. In summary, the results of the present study emphasize that the MT1-MMP-CD44 axis has a unique involvement in T1D development. Accordingly, we suggest that a potent small-molecule MT1-MMP antagonist is required for the design of novel therapies for T1D.
RESUMO
Pathogenesis of type 1 diabetes involves the activation of autoimmune T cells, consequent homing of activated lymphocytes to the pancreatic islets and ensuing destruction of insulin-producing b cells. Interaction between activated lymphocytes and endothelial cells in the islets is the hallmark of the homing process. Initial adhesion, firm adhesion and diapedesis of lymphocytes are the three crucial steps involved in the homing process. Cell-surface receptors including integrins, selectins and hyaluronate receptor CD44 mediate the initial steps of homing. Diapedesis relies on a series of proteolytic events mediated by matrix metalloproteinases. Here, molecular mechanisms governing transendothelial migration of the diabetogenic effector cells are discussed and resulting pharmacological strategies are considered.
Assuntos
Autoimunidade/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/fisiopatologia , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/fisiopatologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/prevenção & controle , Humanos , Hipoglicemiantes/uso terapêuticoRESUMO
The pathogenesis of type 1 diabetes begins with the activation of autoimmune T killer cells and is followed by their homing into the pancreatic islets. After penetrating the pancreatic islets, T cells directly contact and destroy insulin-producing beta cells. This review provides an overview of the dynamic interactions which link T cell membrane type-1 matrix metalloproteinase (MT1-MMP) and the signaling adhesion CD44 receptor with T cell transendothelial migration and the subsequent homing of the transmigrated cells to the pancreatic islets. MT1-MMP regulates the functionality of CD44 in diabetogenic T cells. By regulating the functionality of T cell CD44, MT1-MMP mediates the transition of T cell adhesion to endothelial cells to the transendothelial migration of T cells, thus, controlling the rate at which T cells home into the pancreatic islets. As a result, the T cell MT1-MMP-CD44 axis controls the severity of the disease. Inhibition of MT1-MMP proteolysis of CD44 using highly specific and potent synthetic inhibitors, which have been clinically tested in cancer patients, reduces the rate of transendothelial migration and the homing of T cells. Result is a decrease in the net diabetogenic efficiency of T cells and a restoration of beta cell mass and insulin production in NOD mice. The latter is a reliable and widely used model of type I diabetes in humans. Overall, existing experimental evidence suggests that there is a sound mechanistic rationale for clinical trials of the inhibitors of T cell MT1-MMP in human type 1 diabetes patients.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/fisiologia , Metaloproteinases da Matriz/metabolismo , Linfócitos T/fisiologia , Animais , Humanos , Camundongos , RatosRESUMO
BACKGROUND: Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. METHODS: Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. RESULTS: Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. CONCLUSIONS: We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.
Assuntos
Células Secretoras de Insulina/fisiologia , Células Secretoras de Insulina/transplante , Insulina/metabolismo , Animais , Sobrevivência Celular , Diabetes Mellitus Experimental/cirurgia , Vírus da Leucemia Murina de Friend/genética , Genes Reporter , Glucose/metabolismo , Humanos , Hiperglicemia/prevenção & controle , Secreção de Insulina , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Luciferases/genética , Camundongos , Camundongos SCID , Camundongos Transgênicos , Transplante HeterólogoRESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1-15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1-15 MBP fragment presented in the MHC H-2(U) context. CONCLUSIONS/SIGNIFICANCE: In sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs.