RESUMO
Platelets are specialized cells essential for hemostasis that also function as crucial effectors capable of mediating inflammatory and immune responses. These sentinels continually survey their environment and discriminate between homeostatic and danger signals such as modified components of the extracellular matrix. The glycosaminoglycan hyaluronan (HA) is a major extracellular matrix component that coats the vascular lumen and, under normal conditions, restricts access of inflammatory cells. In response to tissue damage, the endothelial HA matrix enhances leukocyte recruitment and regulates the early stages of the inflammatory response. We have shown that platelets can degrade HA from the surface of activated endothelial cells via the enzyme hyaluronidase-2 (HYAL2) and that HYAL2 is deficient in platelets isolated from patients with inflammatory bowel disease (IBD). Platelets are known to be involved in the pathogenesis of several chronic disease states, including IBD, but they have been largely overlooked in the context of intestinal inflammation. We therefore wanted to define the mechanism by which platelet HYAL2 regulates the inflammatory response during colitis. In this study, we provide evidence that HA catabolism is disrupted in human intestinal microvascular endothelial cells isolated from patients with IBD. Furthermore, mice deficient in HYAL2 are more susceptible to an acute model of colitis, and this increased susceptibility is abrogated by transfusion of HYAL2-competent platelets. Finally, we show that platelets, via HYAL2-dependent degradation of endothelial HA, regulate the early stages of inflammation in colitis by limiting leukocyte extravasation.
Assuntos
Plaquetas/imunologia , Colite/imunologia , Hialuronoglucosaminidase/imunologia , Animais , Plaquetas/patologia , Células Cultivadas , Colite/patologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Proteínas Ligadas por GPI/imunologia , Humanos , Ácido Hialurônico/imunologia , Inflamação/imunologia , Inflamação/patologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Camundongos , Camundongos KnockoutRESUMO
Hyaluronan is the predominant glycosaminoglycan component of the extracellular matrix with an emerging role in hematopoiesis. Modulation of hyaluronan polymer size is responsible for its control over cellular functions, and the balance of hyaluronan synthesis and degradation determines its molecular size. Although two active somatic hyaluronidases are expressed in mammals, only deficiency in hyaluronidase-2 (Hyal-2) results in thrombocytopenia of unknown mechanism. Our results reveal that Hyal-2 knockout mice accumulate hyaluronan within their bone marrow and within megakaryocytes, the cells responsible for platelet generation. Proplatelet formation by Hyal-2 knockout megakaryocytes was disrupted because of abnormal formation of the demarcation membrane system, which was dilated and poorly developed. Importantly, peptide-mediated delivery of exogenous hyaluronidase rescued deficient proplatelet formation in murine and human megakaryocytes lacking Hyal-2. Together, our data uncover a previously unsuspected mechanism of how hyaluronan and Hyal-2 control platelet generation.
Assuntos
Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Megacariócitos/metabolismo , Trombopoese/fisiologia , Animais , Apoptose/fisiologia , Humanos , Immunoblotting , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human ß-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human ß-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hß D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.
Assuntos
Ácido Hialurônico/farmacologia , Imunidade Inata/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Leite Humano/química , Administração Oral , Animais , Anticorpos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Contagem de Colônia Microbiana , Resistência à Doença/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HT29 , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/administração & dosagem , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Lactação/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Período Pós-Parto , Transporte Proteico/efeitos dos fármacos , Salmonelose Animal/imunologia , Salmonelose Animal/patologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Homologia de Sequência de Aminoácidos , Receptor 4 Toll-Like/metabolismo , beta-Defensinas/metabolismoRESUMO
Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human ß-defensin 2 (HßD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HßD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HßD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HßD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HßD2 protein.
Assuntos
Colo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Mucosa Intestinal/metabolismo , beta-Defensinas/biossíntese , Animais , Linhagem Celular Tumoral , Colo/imunologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/imunologia , Ácido Hialurônico/metabolismo , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Imunidade Inata/imunologia , Mucosa Intestinal/imunologia , Camundongos , Camundongos Mutantes , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , beta-Defensinas/genética , beta-Defensinas/imunologiaRESUMO
In addition to mesenchymal cells, endothelial cells may contribute to fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). We investigated whether human intestinal microvascular endothelial cells (HIMEC) undergo EndoMT and contribute to fibrosis in human and experimental inflammatory bowel disease (IBD). HIMEC were exposed to TGF-ß1, IL-1ß, and TNF-α or supernatants of lamina propria mononuclear cells (LPMC) and evaluated for morphological, phenotypic, and functional changes compatible with EndoMT. Genomic analysis was used to identify transcription factors involved in the transformation process. Evidence of in situ and in vivo EndoMT was sought in inflamed human and murine intestine. The combination of TGF-ß1, IL-1ß and TNF-α, or activated LPMC supernatants induced morphological and phenotypic changes consistent with EndoMT with a dominant effect by IL-1. These changes persisted after removal of the inducing agents and were accompanied by functional loss of acetylated LDL-uptake and migratory capacity, and acquisition of de novo collagen synthesis capacity. Sp1 appeared to be the main transcriptional regulator of EndoMT. EndoMT was detected in microvessels of inflammatory bowel disease (IBD) mucosa and experimental colonic fibrosis of Tie2-green fluorescent protein (GFP) reporter-expressing mice. In conclusion, chronic inflammation induces transdifferentiation of intestinal mucosal microvascular cells into mesenchymal cells, suggesting that the intestinal microvasculature contributes to IBD-associated fibrosis through the novel process of EndoMT.
Assuntos
Transdiferenciação Celular/fisiologia , Citocinas/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/patologia , Doenças Inflamatórias Intestinais/patologia , Mesoderma/patologia , Animais , Movimento Celular/fisiologia , Transdiferenciação Celular/genética , Células Cultivadas , Colite/patologia , Colágeno Tipo I/metabolismo , Regulação para Baixo , Matriz Extracelular/metabolismo , Feminino , Fibrose , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Microvasos/patologia , Fenótipo , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Inflammatory bowel disease is a chronic inflammatory condition of the intestinal mucosa whose etiology is unclear but is likely to be multifactorial. We have shown previously that an increased amount of hyaluronan (HA) is present both in the inflamed mucosa of inflammatory bowel disease patients and in isolated human cells after polyI:C treatment. The signal transducer and activator of transcription (STAT)1 protein plays an important role in many signaling pathways that are associated with inflammation. We therefore investigated the role of STAT1 in adhesive interactions that occur between leukocytes and polyI:C-induced mucosal smooth muscle cells (M-SMCs). Activation of STAT1 was observed after the polyI:C treatment of M-SMCs. Specific phosphorylation of tyrosine and serine residues of STAT1 was observed in polyI:C-treated, but not untreated, M-SMC cultures. To evaluate further the role of STAT1, a corresponding STAT-1-null mouse was used. PolyI:C-induced, HA-mediated leukocyte adhesion to colon SMCs from STAT1-null mice was significantly decreased compared with that from wild-type control mice. In vivo, using the dextran sulfate sodium-induced model of colon inflammation, both tissue damage and HA deposition were attenuated in STAT1-null mice compared with that in wild-type control mice. Additionally, the inter-alpha-trypsin inhibitor (IalphaI), a proteoglycan essential for facilitating leukocyte binding to the HA matrix, was reduced in STAT1-null mice. Together, these results demonstrate that STAT1 plays an important role in HA-mediated inflammatory processes.
Assuntos
Adesão Celular , Colite/patologia , Colite/prevenção & controle , Ácido Hialurônico/metabolismo , Leucócitos/citologia , Fator de Transcrição STAT1/deficiência , Animais , Adesão Celular/efeitos dos fármacos , Colite/induzido quimicamente , Colite/metabolismo , Sulfato de Dextrana , Humanos , Mucosa Intestinal/citologia , Leucócitos/efeitos dos fármacos , Camundongos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Poli I-C/farmacologia , Fator de Transcrição STAT1/metabolismo , Células U937RESUMO
Background. Hyaluronan (HA) is a naturally occurring glycosaminoglycan polymer produced in all vertebrates, and usually present at the high molecular weight (>106 Da). Low molecular weight HA has signaling properties, and fragments ~35 kDa size (HA35) have biological activity in eliciting epithelial ß-defensins and tight junction proteins, notably ZO1, important components of innate host defense arsenal of the gut barrier in preclinical models. Safety, tolerability, impact on metabolism, gut permeability, and microbiome composition in healthy human subjects were all evaluated prospectively. Methods. Pharmaceutical grade HA35 (140 mg in water once daily for seven days), was administered orally to 20 healthy subjects (30.7 ± 5.6 years). Demographical, clinical, biochemical laboratory tests, metabolic function and stool microbiome composition were measured on Day 0, 8 and 28. Results. HA35 was tolerated well in all subjects with no serious adverse events in any subjects. No statistical differences in any of the measurements were seen among the study group over the course of the trial. In aggregate there were no changes in demographical, clinical, biochemical laboratory tests, and metabolic function or microbiome composition during the 28-day study. Conclusion. Oral HA35 administration (140 mg/day) is a safe treatment in healthy individuals and does not affect metabolic, inflammatory or microbiome parameters.
Assuntos
Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/efeitos adversos , Adulto , Anti-Inflamatórios , Doenças do Sistema Digestório/induzido quimicamente , Metabolismo Energético , Fezes/química , Fezes/microbiologia , Feminino , Humanos , Ácido Hialurônico/química , Complexo Antígeno L1 Leucocitário/análise , Masculino , Microbiota , Peso Molecular , Projetos Piloto , Estudos Prospectivos , beta-Defensinas/análiseRESUMO
Maintenance of normal blood pressure is critical for preserving the integrity of the cardiovascular system. Angiotensin 1-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-aldosterone system (RAAS) and the renal tubuloglomerular feedback response. Although the two structurally related isozymic forms of ACE both generate the vasoactive octapeptide angiotensin II (Ang II) with equal efficiency, both are expressed in a nonoverlapping tissue-restricted fashion. To discriminate the precise physiological role of each ACE in its requisite tissue in vivo, we expressed one ACE isoform exclusively in a single cell type of an Ace null mouse. Previously, we demonstrated that vascular endothelial cell-specific expression of transgenic somatic ACE (sACE) could restore normal blood pressure of Ace-null mice. In this current study, we expressed germinal ACE (gACE) in the vascular endothelial cells of the Ace null mouse. These mice exhibited correct renal structure, renal function, and normal growth rates. Although the mice had elevated levels of gACE bound to vascular endothelial cells and high levels of gACE and Ang II in the circulating serum, blood pressure was restored only partially. This study demonstrated that gACE, even when expressed in the vasculature, could not functionally substitute for sACE.
Assuntos
Pressão Sanguínea/genética , Peptidil Dipeptidase A/fisiologia , Angiotensina II/sangue , Animais , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peptidil Dipeptidase A/genéticaRESUMO
Intestinal epithelium plays a critical role in host defense against orally acquired pathogens. Dysregulation of this protective barrier is a primary driver of inflammatory bowel diseases (Crohn's and ulcerative colitis) and also infant gastrointestinal infections. Previously, our lab reported that hyaluronan (HA) isolated from human milk induces the expression of the antimicrobial peptide ß-defensin in vivo and protects against Salmonella Typhimurium infection of epithelial cells in vitro. In addition, we demonstrated that commercially available 35 kDa size HA induces the expression of ß-defensin, upregulates the expression of tight junction protein zonula occludens-1 (ZO-1), and attenuates murine Citrobacter rodentium infection in vivo. In this current study, we report that HA35 remains largely intact and biologically active during transit through the digestive tract where it directly induces ß-defensin expression upon epithelial cell contact. We also demonstrate HA35 abrogation of murine Salmonella Typhimurium infection as well as downregulation of leaky tight junction protein claudin-2 expression. Taken together, we propose a dual role for HA in host innate immune defense at the epithelial cell surface, acting to induce antimicrobial peptide production and also block pathogen-induced leaky gut. HA35 is therefore a promising therapeutic in the defense against bacterially induced colitis in compromised adults and vulnerable newborns.
Assuntos
Antibacterianos/uso terapêutico , Ácido Hialurônico/uso terapêutico , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Infecções por Salmonella/tratamento farmacológico , Salmonella typhimurium/efeitos dos fármacos , Animais , Antibacterianos/farmacocinética , Claudina-2/análise , Colo/microbiologia , Colo/patologia , Trânsito Gastrointestinal , Humanos , Ácido Hialurônico/farmacocinética , Imunidade Inata/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Infecções por Salmonella/imunologia , Infecções por Salmonella/patologia , Salmonella typhimurium/imunologia , beta-Defensinas/análiseRESUMO
Tight junction proteins are critical in maintaining homeostatic intestinal permeability. Multiple intestinal inflammatory diseases are correlated with reduced expression of tight junction proteins. We have recently reported that oral treatment of mice with Hyaluronan 35kDa (HA35) increases colonic expression of tight junction protein zonula occludens-1 (ZO-1). Here, we investigate whether HA35 treatment enhances ZO-1 expression by direct interaction with intestinal epithelium in vitro and have identified the HA receptor responsible for HA35-mediated ZO-1 induction in colonic epithelium in vitro and in vivo. Our results reveal that HA35 treatment increases ZO-1 expression in mouse intestinal epithelial organoids, while large HA 2000kDa is not internalized into the cells. Our immunofluorescence data indicate that layilin, but neither toll-like receptor-4 (TLR-4) nor CD44, mediate the HA35-induced ZO-1 expression in colonic epithelium in vitro and in vivo. Additionally, using layilin null mice we have determined that layilin mediates HA35 induction of ZO-1 in healthy mice and during dextran sulfate sodium (DSS)-induced colitis. Furthermore, we find that while ZO-1 expression levels are reduced, layilin expression levels are equivalent in inflammatory bowel disease (IBD) patients and non-IBD controls. Together, our data suggest that layilin is an important HA receptor, that mediates the effect of oral HA35 treatment on intestinal epithelium. HA35 holds promise as a simple dietary supplement to strengthen gut barrier defense.
Assuntos
Proteínas de Transporte/metabolismo , Colite/metabolismo , Ácido Hialurônico/farmacologia , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Camundongos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismoRESUMO
Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection.
Assuntos
Colite/metabolismo , Infecções por Enterobacteriaceae/prevenção & controle , Ácido Hialurônico/administração & dosagem , Mucosa Intestinal/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Administração Oral , Animais , Citrobacter rodentium/efeitos dos fármacos , Colite/induzido quimicamente , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/metabolismo , Regulação da Expressão Gênica , Ácido Hialurônico/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Hyaluronan is an abundant extracellular matrix component prevalent in the vertebrate intestinal tract. Here we discuss what is known about hyaluronan distribution during homeostasis and inflammatory diseases of the gut and discuss ways in which this glycosaminoglycan can participate in regulating innate host defense mechanisms. These natural responses include mechanisms promoting rapid leukocyte recruitment after bacterial challenge/colon tissue damage as well as promoting epithelial defense mechanisms in the intestine.
RESUMO
Hyaluronan (HA) overproduction is a hallmark of multiple inflammatory diseases, including inflammatory bowel disease (IBD). Hyaluronan can act as a leukocyte recruitment molecule and in the most common mouse model of intestinal inflammation, the chemically induced dextran sodium sulfate (DSS) experimental colitis model, we previously determined that changes in colon distribution of HA occur before inflammation. Therefore, we hypothesized that, during a pathologic challenge, HA promotes inflammation. In this study, we tested the progression of inflammation in mice null for the hyaluronan synthase genes (HAS1, HAS3, or both HAS1 and HAS3) in the DSS-colitis model. Our data demonstrate that both the HAS1/HAS3 double and the HAS3 null mice are protected from colitis, compared to wild-type and HAS1 null mice, as determined by measurement of weight loss, disease activity, serum IL-6 levels, histologic scoring, and immunohistochemistry. Most notable is the dramatic increase in submucosal microvasculature, hyaluronan deposition, and leukocyte infiltration in the inflamed colon tissue of wild-type and HAS1 null mice. Our data suggest, HAS3 plays a crucial role in driving gut inflammation. Developing a temporary targeted therapeutic intervention of HAS3 expression or function in the microcirculation may emerge as a desirable strategy toward tempering colitis in patients undergoing flares of IBD.
RESUMO
The 9-2 isozyme of 2-5 (A) synthetase has cellular proapoptotic functions that are mediated not by enzyme activity but by the Bcl-2 homology domain 3 present in its unique carboxyl-terminal region. Another proapoptotic cellular protein is Bax, whose absence in the Bax(-/-) mice causes male sterility due to abnormal sperm differentiation. In this study, we examined whether transgenic 9-2 expression can substitute for the in vivo reproductive function of Bax. To achieve this goal, a sperm-specific promoter was used to drive the expression of 9-2 in the sperm of transgenic mice. By selective cross-breeding, the transgene was transferred to Bax(-/-) mice to generate the experimental mouse line (Bax(-/-), 9-2(+/+)). The male experimental mice were sterile, and their testes maintained the structural abnormality found in Bax(-/-) mice. Thus, the male reproduction functions of Bax could not be replaced by the 9-2 isozyme of 2-5 (A) synthetase.
Assuntos
2',5'-Oligoadenilato Sintetase/fisiologia , Apoptose/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Espermatozoides/citologia , Espermatozoides/enzimologia , 2',5'-Oligoadenilato Sintetase/genética , Animais , Apoptose/genética , Fusão Gênica Artificial/métodos , Cruzamentos Genéticos , Feminino , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Fosfoglicerato Quinase/genética , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Espermatozoides/fisiologia , Proteína X Associada a bcl-2RESUMO
Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.
Assuntos
Pressão Sanguínea/fisiologia , Células Endoteliais/metabolismo , Rim/fisiopatologia , Peptidil Dipeptidase A/metabolismo , Angiotensina II/metabolismo , Animais , Southern Blotting , Creatinina/sangue , Imuno-Histoquímica , Rim/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peptidil Dipeptidase A/genética , Renina/sangueRESUMO
In the latter half of the 20th century, societal and technological changes led to a shift in the composition of the American diet to include a greater proportion of processed, pre-packaged foods high in fat and carbohydrates, and low in dietary fiber (a "Western diet"). Over the same time period, there have been parallel increases in Salmonella gastroenteritis cases and a broad range of chronic inflammatory diseases associated with intestinal dysbiosis. Several polysaccharide food additives are linked to bacterially-driven intestinal inflammation and may contribute to the pathogenic effects of a Western diet. Therefore, we examined the effect of a ubiquitous polysaccharide food additive, maltodextrin (MDX), on clearance of the enteric pathogen Salmonella using both in vitro and in vivo infection models. When examined in vitro, murine bone marrow-derived macrophages exposed to MDX had altered vesicular trafficking, suppressed NAPDH oxidase expression, and reduced recruitment of NADPH oxidase to Salmonella-containing vesicles, which resulted in persistence of Salmonella in enlarged Rab7+ late endosomal vesicles. In vivo, mice consuming MDX-supplemented water had a breakdown of the anti-microbial mucous layer separating gut bacteria from the intestinal epithelium surface. Additionally, oral infection of these mice with Salmonella resulted in increased cecal bacterial loads and enrichment of lamina propria cells harboring large Rab7+ vesicles. These findings indicate that consumption of processed foods containing the polysaccharide MDX contributes to suppression of intestinal anti-microbial defense mechanisms and may be an environmental priming factor for the development of chronic inflammatory disease.
Assuntos
Carboidratos da Dieta/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Viabilidade Microbiana/efeitos dos fármacos , Polissacarídeos/farmacologia , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/fisiologia , Animais , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , NADPH Oxidases/metabolismo , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Angiotensin-converting enzyme (ACE) null mice display aberrant renal pathology. Inadequate formation of angiotensin II (Ang II) results in hypotension, loss of fluid homeostasis, lack of urine concentration, and failure to regulate GFR through the tubuloglomerular feedback (TGF) mechanism. For examining the tissue-specific role of ACE in renal structure and regulation of renal filtrate formation, single-nephron GFR, proximal tubular fluid reabsorption, and TGF responsiveness were determined in mice that expressed ACE in only one tissue. Maximum TGF responses in mice that expressed somatic ACE (sACE) in proximal tubule cells (Gs strain) or germinal ACE in the serum (Pg strain) were reduced significantly compared with wild-type (WT) mice. In contrast, TGF responses in mice that expressed sACE in vascular endothelial cells (Ts strain) were not different from control. Single-nephron GFR was reduced in Ts compared with WT mice, but fractional reabsorption and therefore glomerulotubular balance were not distinguishable. BP responses to exogenous Ang I were diminished in Ts, Gs, and Pg mice, whereas those to Ang II were the same in the different strains. Plasma and renal tissue Ang I of all transgenic mouse strains was significantly higher than WT, whereas Ang II levels were generally lower; aldosterone levels were significantly lower than WT in Ts mice but not in the two other transgenic strains. Our results demonstrate that vascular expression of sACE can largely but not completely restore TGF regulation of GFR. Proximal fluid reabsorption in the chronic absence of proximal tubule ACE is normal.
Assuntos
Angiotensina II/farmacologia , Angiotensina I/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Néfrons/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Aldosterona/análise , Aldosterona/metabolismo , Animais , Determinação da Pressão Arterial , Modelos Animais de Doenças , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Imuno-Histoquímica , Injeções Intravenosas , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Néfrons/fisiologia , Especificidade da EspécieRESUMO
Unlike other RNA polymerases, 2'-5' oligoadenylate synthetases, a family of interferon-induced enzymes, catalyze the formation of 2'-5', not 3'-5', phosphodiester bonds. Moreover, to be active, these proteins require double-stranded RNA as a cofactor. We have been identifying the specific residues of these proteins that impart their novel properties. Here, we report the identity of three such residues that underwent natural mutations in a transgenic mouse line. When deliberately introduced into recombinant proteins, each of these mutations rendered the protein enzymatically inactive. In an effort to understand the roles of these residues in enzyme activity, new mutants carrying other residues in one of these three sites were generated. Detailed characterization of the properties of the mutant proteins revealed that Lys 404 is needed for proper binding of the acceptor substrate, Pro 500 provides structural flexibility to the protein, and Ser 471 is probably required for its proper folding. This study illustrates the power of using natural mutations in transgenes as guides for studying structure-function relationships of proteins.
Assuntos
2',5'-Oligoadenilato Sintetase/química , 2',5'-Oligoadenilato Sintetase/genética , Lisina/genética , Prolina/genética , Serina/genética , Transgenes , Sequência de Aminoácidos , Animais , Ativação Enzimática/genética , Glicina/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
The angiotensin-converting enzyme (ACE) gene encodes two structurally related isozymes, somatic ACE and germinal ACE, that are uniquely expressed in discrete locations in the body. The importance of ACE in these cell types was revealed by generating Ace -/- mice, which exhibit multiple abnormalities including renal structural defects and functions, hypotension, and male sterility. To test the hypothesis that specific physiological functions of ACE are mediated by isozyme-specific and tissue-specific expression patterns, we have used a transgenic approach to develop mouse strains that express just one ACE isoform in the target tissue of Ace -/- mice. The mice described in this report produce germinal ACE in sperm and serum. These mice were as healthy as wild type mice, and the males were fertile. Interestingly, they had normal kidney structure, fluid homeostasis, and partially restored urine concentration despite having low blood pressure. This result demonstrated that circulating germinal ACE is sufficient for maintaining normal kidney structure and fluid homeostasis but insufficient for restoring blood pressure to normal levels.
Assuntos
Isoenzimas/metabolismo , Rim/fisiologia , Peptidil Dipeptidase A/metabolismo , Animais , Isoenzimas/genética , Rim/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , Peptidil Dipeptidase A/genética , TransgenesRESUMO
Angiotensin-converting enzyme (ACE) is expressed in many tissues, including vasculature and renal proximal tubules, and its genetic ablation in mice causes abnormal renal structure and functions, hypotension, and male sterility. To test the hypothesis that specific physiological functions of ACE are mediated by its expression in specific tissues, we generated different mouse strains, each expressing ACE in only one tissue. Here, we report the properties of two such strains of mice that express ACE either in vascular endothelial cells or in renal proximal tubules. Because of the natural cleavage secretion process, both groups also have ACE in the serum. Both groups were as healthy as wild-type mice, having normal kidney structure and fluid homeostasis, though males remained sterile, because they lack ACE expression in sperm. Despite equivalent serum ACE and angiotensin II levels and renal functions, only the group that expressed ACE in vascular endothelial cells had normal blood pressure. Expression of ACE, either in renal proximal tubules or in vasculature, is sufficient for maintaining normal kidney functions. However, for maintaining blood pressure, ACE must be expressed in vascular endothelial cells. These results also demonstrate that ACE-mediated blood pressure maintenance can be dissociated from its role in maintaining renal structure and functions.