Role of cystatin C in urogenital malignancy.

Urogenital malignancy accounts for one of the major causes of cancer-related deaths globally. Numerous studies have investigated novel molecular markers in the blood circulation, tumor tissue, or urine in order to assist in the clinical identification of tumors at early stages, predict the response of therapeutic strategies, and give accurate prognosis assessment. As an endogenous inhibitor of lysosomal cysteine proteinases, cystatin C plays an integral role in diverse processes. A substantial number of studies have indicated that it may be such a potential promising biomarker. Therefore, this review was intended to provide a detailed overview of the role of cystatin C in urogenital malignancy.

Proteins linked to atherosclerosis and cell proliferation are associated with the shrunken pore syndrome in heart failure patients: Shrunken pore syndrome and proteomic associations.

PURPOSE: The "Shrunken pore syndrome" (SPS) is characterized by a difference in renal filtration between cystatin C and creatinine, resulting in a low eGFRcystatinC /eGFRcreatinine -ratio. Studies have demonstrated a high risk for cardiovascular morbidity and mortality for patients with SPS. In this discovery study, we explored associations between SPS and proteins implicated in cardiovascular disease and inflammation in patients with heart failure. EXPERIMENTAL DESIGN: Plasma samples from 300 individuals in HARVEST-Malmö trial hospitalized for the diagnosis of heart failure (mean age 74.9 ± 11.5 years; 30.0% female), were analyzed with a proximity extension assay consisting of 92 proteins. A Bonferroni-corrected p-value of 0.05/92 = 5.4 × 10-4 was considered significant in the initial age and sex-adjusted analyses. Presence of SPS was defined as eGFRcystatinC ≤ 60% of eGFRcreatinine . RESULTS: SPS presented with significant associations (p < 5.4 × 10-4 ) in age and sex-adjusted logistic regressions with elevated levels of six proteins; scavenger receptor cysteine rich type 1 protein M130, tumor necrosis factor receptor 1, tumor necrosis factor receptor 2, osteoprotegerin, interleukin-2 receptor subunit alpha, and tyrosine-protein kinase receptor UFO. All proteins remained associated (p < 0.05) with SPS after multivariate adjustments. CONCLUSIONS AND CLINICAL RELEVANCE: In heart failure patients, SPS was associated with proteins linked to atherosclerosis and cell proliferation.

Suppressed dendritic cell functions by cystatin C lead to compromised immunity in vivo.

Pathogenic microorganisms utilize multiple approaches to break down host immunity in favor of their invasion, of which, cystatin C is one of the soluble factors secreted by parasites reported to affect host immunity in vivo. The cellular targets and mechanisms of action in vivo of cystatin C, however, are far from clear. As professional antigen-presenting cells, dendritic cells (DCs) are first immune cells that contact foreign pathogenic agents or their products to initiate immune responses. We previously reported that cystatin C can regulate the functions of DCs in terms of suppressed CD4+ T cell activation but enhanced Th1/Th17 differentiation via different mechanisms. Here, we further verified these regulatory effects of cystatin C on DCs in vivo. We found that the suppressive role of DC-mediated CD4+ T cell proliferation by cystatin C was partly cell-contact independent and extended to CD8+ T cells in vivo. Although cystatin C-overexpressing DCs trafficked equally as their mock-transduced counterparts, their adoptive transfer suppressed CD8+ T cell immunity and resulted in compromised tumor rejection in both vaccination and treatment regimes. Compared with their role in promoting Th17 differentiation in vivo, cystatin C-transduced DCs had far greater ability to induce T regulatory cells (Tregs), leading to collectively a higher Treg/Th17 ratio in an adoptively transferred disease model, and thus relieved Th17-dependent autoimmunity. Collectively, these data demonstrated strong in vivo evidences for immune regulatory roles of cystatin C in DCs and provided theoretical basis for the application of cystatin C-transduced cell therapy in the treatment or remission of certain autoimmune diseases. (246).

Increased Rate of Retinal Pigment Epithelial Cell Migration and Pro-Angiogenic Potential Ensuing From Reduced Cystatin C Expression.

Purpose: Variant B precursor cysteine protease inhibitor cystatin C, a known recessive risk factor for developing exudative age-related macular degeneration (AMD), presents altered intracellular trafficking and reduced secretion from retinal pigment epithelial (RPE) cells. Because cystatin C inhibits multiple extracellular matrix (ECM)-degrading cathepsins, this study evaluated the role of this mutation in inducing ECM-related functional changes in RPE cellular behavior. Methods: Induced pluripotent stem cells gene-edited bi-allelically by CRISPR/Cas9 to express the AMD-linked cystatin C variant were differentiated to RPE cells and assayed for their ability to degrade fluorescently labeled ECM proteins. Cellular migration and adhesion on multiple ECM proteins, differences in transepithelial resistance and polarized protein secretion were tested. Vessel formation induced by gene edited cells-conditioned media was quantified using primary human dermal microvascular epithelial cells. Results: Variant B cystatin C-expressing induced pluripotent stem cells-derived RPE cells displayed a significantly higher rate of laminin and fibronectin degradation 3 days after seeding on fluorescently labeled ECM (P < 0.05). Migration on matrigel, collagen IV and fibronectin was significantly faster for edited cells compared with wild-type (WT) cells. Both edited and WT cells displayed polarized secretion of cystatin C, but transepithelial resistance was lower in gene-edited cells after 6 weeks culture, with significantly lower expression of tight junction protein claudin-3. Media conditioned by gene-edited cells stimulated formation of significantly longer microvascular tubes (P < 0.05) compared with WT-conditioned media. Conclusions: Reduced levels of cystatin C lead to changes in the RPE ability to degrade, adhere, and migrate supporting increased invasiveness and angiogenesis relevant for AMD pathology.

Cystatin C serum levels in healthy children are related to age, gender, and pubertal stage.

BACKGROUND: This study aims to establish age- and gender-specific cystatin C (CysC) reference values for healthy infants, children, and adolescents and to relate them to pubertal stage, height, weight, and body mass index (BMI). METHODS: Serum CysC and creatinine levels of 6217 fasting, morning venous blood samples from 2803 healthy participants of the LIFE Child study (age 3 months to 18 years) were analyzed by an immunoassay. Recruitment started in 2011; 1636 participants provided at least one follow-up measurement. Percentiles for CysC were calculated. Age- and gender-related effects of height, weight, BMI, and puberty status were assessed through linear regression models. RESULTS: Over the first 2 years of life, median CysC levels decrease depending on height (ß = - 0.010 mg/l/cm, p < 0.001) and weight (ß = - 0.033 mg/l/kg, p < 0.001) from 1.06 to 0.88 mg/l for males and from 1.04 to 0.87 mg/l for females. Following the second year of age, the levels remain stable for eight years. From 11 to 14 years of age, there is an increase of median CysC levels in males to 0.98 mg/l and a decrease in females to 0.86 mg/l. The change is associated with puberty (ß = 0.105 mg/l/Tanner stage, p < 0.001 in males and ß = - 0.093 mg/l/Tanner stage, p < 0.01 in females) and in males with height (ß = 0.003 mg/l/cm, p < 0.001). CONCLUSIONS: CysC levels depend on age, gender, and height, especially during infancy and puberty. We recommend the use of age- and gender-specific reference values for CysC serum levels for estimating kidney function in clinical practice.

On the role of cystatin C in cancer progression.

Cystatin C (Cyst C) is an endogenous inhibitor of lysosomal cysteine proteinases, which has been shown to play a role in several normal and pathological processes. Interestingly, a growing number of experimental and clinical studies suggest that this inhibitor also appears to be implicated in the malignant progression of various human tumors. However, the role of Cyst C in malignant diseases is still controversial as these studies have highlighted that this protein may function either as tumor suppressor or tumor promoter. The specific mechanisms underlying these opposing effects at present remain murky and are the subject of many current investigations. On the other hand, a complete knowledge of these mechanisms is of clinical interest in order to develop new, effective antitumor treatments based on the appropriate use of natural and/or synthetic cysteine proteinase inhibitors. This paper discusses the current findings regarding the role of Cyst C in cancer progression and the clinical implications emerging from these studies.

A new insight into cystatin C contained in milk basic protein to bone metabolism: Effects on osteoclasts and osteoblastic MC3T3-E1 cells in vitro.

A milk protein fraction possessing alkaline isoelectric points (milk basic protein [MBP]) improves bone metabolism in vivo, and it inhibits bone resorption by osteoclasts and promotes mouse osteoblastic MC3T3-E1 cells proliferation in vitro. Cystatin C (CysC) is a component of MBP and shows bone resorption inhibitory activity. Therefore, it is likely that MBP with higher CysC content improves bone metabolism more effectively. In this study, we prepared MBP with low and high contents of CysC and compared its effects on bone metabolism with standard MBP in vitro. Our results showed that the CysC content in MBP was positively related to not only bone resorption inhibitory activity but also MC3T3-E1 cells proliferative activity. Furthermore, purified CysC stimulated MC3T3-E1 cells proliferation. These results indicate that CysC contributes to promotion of MC3T3-E1 cells proliferation, and MBP with higher CysC content shows enhanced bone resorption inhibitory activity and MC3T3-E1 cells proliferative activity. CysC is considered an important factor in the effect on bone metabolism of MBP.

Expression of cystatin C in the female reproductive tract and its effect on human sperm capacitation.

BACKGROUND: Cystatin C (CST3), a cysteine protease inhibitor in seminal plasma, is expressed in animal uteri. However, its expression in the human female reproductive tract and its effect on human sperm capacitation are unclear. METHODS: The cellular localization of CST3 was observed using immunohistochemistry. The binding of CST3 to sperm was examined using immunocytochemistry. Sperm motility parameters were analyzed using computer-assisted sperm analysis. Sperm capacitation was evaluated by analyzing cholesterol content, protein tyrosine phosphorylation levels, and the acrosome reaction. RESULTS: Immunohistochemical staining demonstrated that CST3 is prominently expressed in the female reproductive tract, including the epithelial lining and cervix and endometrium fluids, particularly at times near ovulation. It can bind to human sperm on the post-acrosomal head region and the mid and principal piece of the tail. CST3 enhances sperm motility and inhibits the signal initiating sperm capacitation, i.e., efflux of cholesterol from the sperm plasma membrane and a late sperm capacitation event, i.e., the increase in the sperm protein tyrosine phosphorylation. The suppressive trend on sperm acrosome reaction further supports CST3's ability to inhibit sperm capacitation. CONCLUSIONS: These findings suggest that cervical CST3 may prevent precocious capacitation and acrosome reaction, thus preserving sperm fertilizing ability before it reaches the fallopian tube. Additionally, CST3 may help sperm enter the upper reproductive tract by enhancing sperm motility.

Adiposity and the Role of Neuroendocrine Hormones in Energy Balance.

Obesity is a worldwide health concern and, given the risk for developing associated co-morbidities that increase mortality, obesity has health implications for individuals and populations. Obesity involves multifactorial regulatory mechanisms, and recognition of these mechanisms will enhance the care critical care nurses provide to their patients. This article reviews the general physiological mechanisms of energy storage and the regulation of adiposity related to key neuroendocrine hormones. The authors also highlight the implications for nursing care for critically ill patients suffering from obesity.

The protease inhibitor cystatin C down-regulates the release of IL-ß and TNF-α in lipopolysaccharide activated monocytes.

Human cystatin C, a member of the cysteine proteinase-inhibitory family, is produced by all nucleated cells and has important roles in regulating natural immunity. Nematode homologs to human cystatin C have been shown to have anti-inflammatory effects on monocytes and to reduce colitis in mice. In Crohn's disease, pathogenic activated monocytes help drive inflammatory processes via the release of proinflammatory cytokines and chemokines. In particular, tumor necrosis factor-α-producing inflammatory monocytes have a central role in the intestinal inflammation in patients with Crohn's disease. We investigated the potential of human cystatin C to regulate pathogenic activated monocytes and its potential as an Immunomodulator in Crohn's disease. We found that cystatin C significantly decreased the lipopolysaccharide-stimulated release and expression of interleukin-1ß and tumor necrosis factor-α in monocyte and peripheral blood mononuclear cell cultures from healthy donors, whereas interleukin-6 and interleukin-8 levels were unchanged. A similar reduction of interleukin-1ß and tumor necrosis factor-α was also seen in peripheral blood mononuclear cell cultures from patients with Crohn's disease, and in particular, tumor necrosis factor-α was reduced in supernatants from lamina propria cell cultures from patients with Crohn's disease. Further investigation revealed that cystatin C was internalized by monocytes via an active endocytic process, decreased phosphorylation of the mitogen-activated protein kinase pathway extracellular signal-regulated kinase-1/2, and altered surface marker expression. The ability of cystatin C to modulate the cytokine expression of monocytes, together with its protease-inhibitory function, indicates that modulation of the local cystatin C expression could be an option in future Crohn's disease therapy.

Cystatin C as a p53-inducible apoptotic mediator that regulates cathepsin L activity.

In response to various cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes. However, the full picture of the p53 downstream pathway still remains to be elucidated. Here we identified cystatin C, a major inhibitor of cathepsins, as a novel p53 target. In response to DNA damage, activated p53 induced cystatin C expression through p53 binding sequence in the first intron. We showed that cathepsin L activity was decreased in HCT116 p53(+/+) cells after adriamycin treatment, but not in HCT116 p53(-/-) cells. We also found that knockdown of cystatin C reduced adriamycin-induced caspase-3 activation. Cystatin C expression was significantly downregulated in breast cancer cells with p53 mutations, and decreased cystatin C expression was associated with poor prognosis of breast cancer. Our findings revealed an important role of the p53-cystatin C pathway in human carcinogenesis.

Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance.

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel lead to viscous secretions from submucosal glands that cannot be properly hydrated and cleared by beating cilia in cystic fibrosis (CF) airways. The mechanisms by which CFTR, and the predominant epithelial sodium channel (ENaC), control the hydration and clearance of glandular secretions remain unclear. We used a proteomics approach to characterize the proteins contained in CF and non-CF submucosal gland fluid droplets and found that differentially regulated proteases (cathepsin S and H) and their antiprotease (cystatin C) influenced the equilibration of fluid on the airway surface and tracheal mucociliary clearance (MCC). Contrary to prevailing models of airway hydration and clearance, cystatin C, or raising the airway surface liquid (ASL) pH, inhibited cathepsin-dependent ENaC-mediated fluid absorption and raised the height of ASL, and yet decreased MCC velocity. Importantly, coupling of both CFTR and ENaC activities were required for effective MCC and for effective ASL height equilibration after volume challenge. Cystatin C-inhibitable cathepsins controlled initial phases of ENaC-mediated fluid absorption, whereas CFTR activity was required to prevent ASL dehydration. Interestingly, CF airway epithelia absorbed fluid more slowly owing to reduced cysteine protease activity in the ASL but became abnormally dehydrated with time. Our findings demonstrate that, after volume challenge, pH-dependent protease-mediated coupling of CFTR and ENaC activities are required for rapid fluid equilibration at the airway surface and for effective MCC. These findings provide new insights into how glandular fluid secretions may be equilibrated at the airway surface and how this process may be impaired in CF.

Cystatin C: a kidney function biomarker.

Renal function is essential for homeostasis. The kidneys play important pleiotropic roles including removal of metabolic waste products and maintenance of water-electrolyte balance and blood pressure. Early diagnosis of renal dysfunction and institution of appropriate therapy are vital to survival. Unfortunately, common indicators of renal function lack necessary sensitivity and specificity. Recent evidence has, however, suggested that cystatin C (cysC) may be useful as a marker for glomerular filtration. CysC is a protein belonging to a group of cysteine proteases inhibitors produced primarily by nucleated cells. Due to low molecular weight and positive pI, it is easily filtered. Moreover, its serum concentration is independent of gender, age, or muscle mass, i.e., typical confounders in assessing glomerular filtration rate (GFR). This chapter discusses the structure and biologic function of cysC, its role as an indicator of GFR, and the most frequently used methods for its determination.

Recombinant human erythropoietin pretreatment attenuates acute renal tubular injury against ischemia-reperfusion by restoring transient receptor potential channel-6 expression and function in collecting ducts.

OBJECTIVE: Acute renal tubular injury is a serious complication in the postoperative period, which is associated with high mortality and increased ICU stay. We aimed to demonstrate the protective effect of rhEPO against acute tubular injury induced by ischemia-reperfusion and to explore the mechanism of canonical transient receptor potential channel-6. DESIGN: Randomized laboratory animal study. SETTINGS: Animal research laboratory. INTERVENTIONS: Male Sprague-Dawley rats were randomly divided into three groups: the sham group, the control group, and the rhEPO group. Experimental acute tubular injury was established in rats by bilateral renal arterial occlusion for 30 minutes followed by reperfusion. MEASUREMENTS AND MAIN RESULTS: Blood samples were obtained for cystatin-C and neutrophil gelatinase-associated lipocalin measurements by enzyme-linked immunosorbance assays. Seventy-two hours after reperfusion, urine samples were collected for osmolality and fractional excretion of sodium (%) assays on a chemistry analyzer. Kidneys were harvested at 24, 48, and 72 hours after reperfusion. Transient receptor potential channel-6, aquaporin-2, and Na,K-ATPase expression in collecting ducts were studied by immunofluorescence and Western blot. Coimmunoprecipitations were also performed to identify the possible signalplex relation between transient receptor potential channel-6 and aquaporin-2 or Na,K-ATPase channels. RhEPO pretreatment significantly inhibited serum cystatin-C (2 hr: 453 ± 64 µg/L vs 337 ± 28 µg/L, p < 0.01), serum neutrophil gelatinase-associated lipocalin (72 hr: 1,175 ± 107 ng/L vs 1,737 ± 402 ng/L, p < 0.05), and urinary fractional excretion of sodium (%) increase (0.9 ± 0.1 vs 2.2 ± 0.8, p < 0.05) and alleviated the decrease of urinary osmolality (1,293 ± 101 mosmol/kg H2O vs 767 ± 91 mosmol/kg H2O, p < 0.05) induced by ischemia-reperfusion injury. Meanwhile, recombinant human erythropoietin greatly improved the ischemia-reperfusion-induced attenuation of transient receptor potential channel-6 expression (48 hr: 42% ± 2% vs 67% ± 2% and 72 hr: 55% ± 2% vs 66% ± 2%), as well as aquaporin-2 and Na,K-ATPase expression in collecting ducts. Transient receptor potential channel-6 functionally interacted with Na,K-ATPase but not aquaporin-2. CONCLUSIONS: Recombinant human erythropoietin pretreatment at the dose of 5,000 IU/kg potently prevented ischemia-reperfusion-induced acute tubular injury, which might be partly attributed to the restoring the effect of transient receptor potential channel-6 expression and collecting duct function.

Regulation of TGF-ß1-driven differentiation of human lung fibroblasts: emerging roles of cathepsin B and cystatin C.

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-ß1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation, and led to an accumulation of intracellular 50-kDa TGF-ß1. Moreover, the addition of Cat B generated a 25-kDa mature form of TGF-ß1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation but had no effect on p38 MAPK and JNK phosphorylation, indicating that Cat B mostly disturbs TGF-ß1-driven canonical Smad signaling pathway. Although mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-ß1-induced differentiation of idiopathic pulmonary fibrosis and CCD-19Lu fibroblasts. In addition, cystatin C participated in the control of extracellular Cats, because its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-ß1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.

Análisis de anticuerpos antifosfolipídicos y cistatina C en pacientes con esclerosis múltiple / Analysis of antiphospholipid antibodies and cystatin C in multiple sclerosis patients / Teste de anticorpos antifosfolípídes e cistatina C em pacientes com esclerose múltipla

La cistatina C es considerada el inhibidor fisiológico más importante de las proteasas de cisteína endógenas. Se cree que el papel de la cistatina C es el de modular la actividad de las proteasas secretadas o liberadas de células dañadas o en proceso de necrosis, siendo por tanto las cistatinas fundamentales para los procesos de regulación y prevención del potencial daño proteolítico local. Los anticuerpos antifosfolípidos se usan para esclarecer el diagnóstico de esclerosis múltiple (EM) ya que existen patologías que pueden cursar con sintomatología o hallazgos paraclínicos semejantes. El objetivo de este trabajo fue analizar la concentración de cistatina C y la presencia o ausencia de anticuerpos antifosfolipídicos en pacientes diagnosticados de esclerosis múltiple remitente recurrente (EMRR) como marcadores de desmielinización. Este trabajo se llevó a cabo conjuntamente por el laboratorio de Riesgo Vascular, el laboratorio de Autoinmunidad y la Unidad de Esclerosis Múltiple del Hospital Universitario Virgen Macarena de Sevilla, España, con una duración de un año. Se seleccionaron dos tipos de poblaciones: grupo 1, n=30 pacientes con EMRR y un segundo grupo, denominado grupo control, n=30. Se determinó cistatina C y anticuerpos antifosfolípidos IgG e IgM, anticuerpos anticardiolipina IgG e IgM y anticuerpos f>2 glicoproteína IgG e IgM. Los pacientes diagnosticados de EMRR presentan títulos negativos de anticuerpos antifosfolípidos IgG e IgM, anticardiolipina IgG e IgM y f>2 glicoproteína IgG e IgM. La concentración de cistatina C es menor en el grupo de pacientes diagnosticados de EM, lo que podría producir un déficit en la modulación de las proteasas de cisteína endógenas. Dicha desmielini-zación agudizaría el progreso de la EM.

Cystatin C is considered the most important physiological inhibitor of endogenous cysteine proteases; the role of cystatin C is believed to be to modulate the activity of proteases secreted or released from damaged cells or in the process of necrosis, therefore cystatins being fundamental regulatory processes and a potential prevention of local proteolytic damage. Antiphospholipid antibodies are used to clarify the diagnosis of diseases like multiple sclerosis (MS) and other pathologies could present similai symptoms or paraclinical findings. The objective of the present work is to analyze the concentration of cystatin C and the presence or absence of antiphospholipid antibodies in patients diagnosed with relapsing remitting multiple sclerosis (RRMS) as markers of demyelization. This work was carried out jointly by the Vascular Risk Laboratory, the Laboratory of Autoimmunity and Multiple Sclerosis Unit, Hospital Universitario Virgen Macarena in Seville in one year. Two types of people were selected: Group 1 (n = 30) RRMS group and a control group, n = 30. Cystatin C and antiphospholipid antibodies IgG and IgM, IgG and IgM anticardiolipin, $2 glycoprotein IgG and IgM were determined. Patients showed negative titers of antiphospholipid antibodies IgG and IgM, IgG and IgM anticardiolipin, $2 glycoprotein IgG and IgM. Cystatin C concentration is lower in the group of patients diagnosed with MS, which could give rise to a decrease in the modulation of endogenous cysteine proteases. This would exacerbate the progress of demyelization in MS.

A cistatina C é considerada o inibidor fisiológico das proteases de cisteína endógenas mais importante. Acredita-se que o papel da cistatina C é o de modular a atividade de proteases secretadas ou liberadas a partir de células danificadas ou em processo de necrose, sendo por isso as cistatinas fundamentais para os processos de regulagao e prevengao do potencial dano proteolítico local. Anticorpos antifosfolípides sao usados para esclarecer o diagnóstico de EM, visto que existem patologias que podem apresentar sintomas ou achados paraclínicos semelhantes. O objetivo deste trabalho foi o de analisar a concentra-gao de cistatina C e a presenga ou ausencia de anticorpos antifosfolípides em pacientes diagnosticados com esclerose múltipla recidivante - remitente (EMRR) como marcadores de desmielinizagao. Este trabalho foi realizado em conjunto pelo laboratório de Risco Vascular, o laboratório de Autoimunidade e a Unidade de Esclerose Múltipla do Hospital Universitario Virgen Macarena, de Sevilha, Espanha, com uma duragao de um ano. Foram selecionados dois tipos de populagdes-. Grupo 1 (n = 30) pacientes com EMRR e um segundo grupo, chamado de grupo controle, n = 30. Determinou-se cistatina C e anticorpos antifosfolípides IgG e IgM, anticorpos anticardiolipina IgG e IgM, e anticorpos $2 glicoproteína IgG e IgM. Pacientes diagnosticados com EMRR apresentam títulos negativos de anticorpos antifosfolípides IgG e IgM, anticardiolipina IgG e IgM e $2 glicoproteína IgG e IgM. A concentragao de cistatina C é menor no grupo de pacientes diagnosticados com EM, o que poderia produzir um déficit na modulagao das proteases de cisteína endógenas. Tal desmielinizagao agravaría o progresso da EM.

Olive flounder (Paralichthys olivaceus) cystatin C: cloning, mRNA expression, and enzymatic characterization of olive flounder cystatin C.

Cystatins are endogenous inhibitors of mammalian lysosomal cysteine proteinases, such as cathepsins B, L, H, and S. Cystatin C belongs to the type 2 cystatin family. In this study, the 751-bp cystatin C cDNA (PoCystatin C) of olive flounder (Paralichthys olivaceus) was cloned by screening from the olive flounder cDNA library. The mRNA expression of the PoCystatin C gene was examined in various tissues from normal and lipopolysaccharide (LPS)-stimulated olive flounder by RT-PCR and was compared with inflammatory cytokines IL-1ß, IL-6, and IL-8. PoCystatin C transcripts ubiquitously existed in all normal and LPS-stimulated tissues that were tested. The recombinant PoCystatin C protein was expressed in Escherichia coli BL21(DE3) in pCold™ TF DNA expression vector as a 70-kDa fusion protein. The protease inhibitory activities of recombinant PoCystatin C toward papain cysteine protease, piscine cathepsins (L, S, K, F, and X), and bovine cathepsin B were measured with the synthetic fluorogenic peptide substrates. PoCystatin C tightly inhibited papain cysteine protease, whereas cathepsins L, S, K, F, X, and B were inhibited with lower affinities. Our results indicate that the P. olivaceus cystatin C is a homolog of mammalian cystatin C due to its sequence, structure, tissue expression, and biochemical activity.

Role of cystatin C in amyloid precursor protein-induced proliferation of neural stem/progenitor cells.

The amyloid precursor protein (APP) is well studied for its role in Alzheimer disease. However, little is known about its normal function. In this study, we examined the role of APP in neural stem/progenitor cell (NSPC) proliferation. NSPCs derived from APP-overexpressing Tg2576 transgenic mice proliferated more rapidly than NSPCs from the corresponding background strain (C57Bl/6xSJL) wild-type mice. In contrast, NSPCs from APP knock-out (APP-KO) mice had reduced proliferation rates when compared with NSPCs from the corresponding background strain (C57Bl/6). A secreted factor, identified as cystatin C, was found to be responsible for this effect. Levels of cystatin C were higher in the Tg2576 conditioned medium and lower in the APP-KO conditioned medium. Furthermore, immunodepletion of cystatin C from the conditioned medium completely removed the ability of the conditioned medium to increase NSPC proliferation. The results demonstrate that APP expression stimulates NSPC proliferation and that this effect is mediated via an increase in cystatin C secretion.

Genetic predisposition to left ventricular hypertrophy and the potential involvement of cystatin-C in untreated hypertension.

BACKGROUND: The angiotensinogen M235T and aldosterone synthase C-344T gene polymorphisms have been associated with cardiac and structure function. However, these associations in untreated hypertension remain unknown. We examined whether these variants determined both echocardiography indices and the potential associated underlying mechanisms, including cystatin-C and vascular inflammation. METHODS: The study population consisted of 319 untreated patients and 191 healthy individuals. Polymorphisms were determined by polymerase chain reaction technique. Left cardiac indices of geometry and function were assessed by echocardiography. Cystatin-C, intracellular cell adhesion molecule 1, and vascular cell adhesion molecule 1 levels were measured by enzyme-linked immunosorbent assay, whereas high sensitivity C-reactive protein levels were measured by immunonephelometry. RESULTS: There was no significant interaction between the angiotensinogen genotypes on left ventricular mass index (LVMI) and diastolic function indices in all study groups. Regarding C-344T polymorphism, TT homozygous hypertensive subjects exhibited higher values of LVMI compared with C allele carriers (P = 0.02) and higher prevalence of concentric hypertrophy (P < 0.001). However, this polymorphism was not associated with variations in left atrial volume and diastolic dysfunction. Cystatin-C levels were correlated with LVMI values (r = 0.22; P = 0.002) and mean E/A ratio (r = -0.24; P < 0.001). Interestingly, a linear increase of LVMI with cyctatin-C quartiles has been revealed (F = 5.01; P < 0.001). Moreover, post hoc tests showed that increased levels of cystatin-C (above 75th percentile) were significantly different between both the first (P = 0.009) and the second quartile (P = 0.02). CONCLUSIONS: We have shown that C-344T potentially predicts higher values of LVMI and concentric hypertrophy in untreated hypertension, independently of renal function and subclinical inflammation. Increased levels of cystatin-C were correlated with higher LVMI values.

Involvement of dopaminergic neuronal cystatin C in neuronal injury-induced microglial activation and neurotoxicity.

Factors released from injured dopaminergic (DA) neurons may trigger microglial activation and set in motion a vicious cycle of neuronal injury and inflammation that fuels progressive DA neurodegeneration in Parkinson's disease. In this study, using proteomic and immunoblotting analysis, we detected elevated levels of cystatin C in conditioned media (CM) from 1-methyl-4-phenylpyridinium and dieldrin-injured rat DA neuronal cells. Immunodepletion of cystatin C significantly reduced the ability of DA neuronal CM to induce activation of rat microglial cells as determined by up-regulation of inducible nitric oxide synthase, production of free radicals and release of proinflammatory cytokines as well as activated microglia-mediated DA neurotoxicity. Treatment of the cystatin C-containing CM with enzymes that remove O- and sialic acid-, but not N-linked carbohydrate moieties markedly reduced the ability of the DA neuronal CM to activate microglia. Taken together, these results suggest that DA neuronal cystatin C plays a role in the neuronal injury-induced microglial activation and neurotoxicity. These findings from the rat DA neuron-microglia in vitro model may help guide continued investigation to define the precise role of cystatin C in the complex interplay among neurons and glia in the pathogenesis of Parkinson's disease.