Human serum albumin fusion protein as therapeutics for targeting amyloid beta in Alzheimer's diseases.

Alzheimer's disease (AD) is characterized by amyloid beta (Aß) plaques and neurofibrillary tangles. AD drug development has been limited due to the presence of the blood-brain barrier (BBB), which prevents efficient uptake of therapeutics into the brain. To solve this problem, we used trans-activator of transcription (TAT)-transducing domain and added the human serum albumin (HSA) carrier to increase the half-life of the drug within the body. In addition, we included the protein of interest for lowering Aß deposition and/or neurofibrillary tangles. We made HSA fusion protein (designated AL04) which contains Cystatin C (CysC) as core mechanism of action moiety in the construct containing tandem repeat TAT (dTAT). After purification of 80KDa AL04, we investigate the therapeutic potential of AL04 in vitro and AD mouse model Tg2576. We evaluated the permeability of AL04 through the BBB using a cell-basedhuman BBB model and show that dTAT plays a role in facilitating the delivery of 80 kDa protein. We found out that AL04 attenuates Aß-induced neurotoxicity in PC12 cells. In Tg2576 mice brain, Aß plaques were dramatically reduced in AL04 treated mice. These data suggest that BBB-crossing albumin fusion protein AL04 with CysC active moiety can be a disease modifying treatment for AD.

Population Pharmacokinetic Analysis and Dosing Optimization Based on Unbound Daptomycin Concentration and Cystatin C in Nonobese Elderly Patients with Hypoalbuminemia and Chronic Kidney Disease.

PURPOSE: This study evaluated the population pharmacokinetics of daptomycin in nonobese elderly patients with hypoalbuminemia and chronic kidney disease (CKD) using the glomerular filtration rate estimated from cystatin C (eGFRcys) and estimated its optimal dose. METHODS: We performed population pharmacokinetic analysis of the unbound concentrations of daptomycin. The probability of target attainment of 90% for achieving an area under the concentration-time curve of unbound daptomycin at steady state/ minimum inhibitory concentration ratio of ≥66.6 was stochastically simulated. RESULTS: In the population pharmacokinetic analysis of 25 patients aged ≥65 years, the two-compartment model using eGFRcys and age as covariates of clearance in central compartment of unbound daptomycin were optimal. The unbound fraction rate (fu) was 0.05-0.14. According to the Monte Carlo simulation, the optimal doses for patients with eGFRcys of 20-60 mL/min and aged 65-95 years were calculated as 200-500 mg q24h. CONCLUSION: These results suggest that establishing the dose using total concentrations may result in under- or overestimation caused by alterations in fu. The optimal dose for nonobese elderly patients with hypoalbuminemia and CKD depends on eGFRcys and age, and a standard dose may be insufficient for some patients.

Cystatin C alleviates H2O2-induced H9c2 cell injury.

OBJECTIVE: At present, the incidence of acute myocardial infarction is increasing year by year, and it has become one of the diseases with the highest mortality rate in humans. Myocardial ischemia-reperfusion injury (MIRI) is a major problem in the treatment of myocardial infarction, but clinically there is no effective way to treat MIRI. This study used Cystatin C (Cys C) to treat cardiomyocytes and rats to investigate the effect of Cys C on MIRI. MATERIALS AND METHODS: We used H2O2 to induce rat cardiomyocytes (H9c2 cells) injury and stimulated the cells with Cys C. Cell counting kit 8 (CCK8) assay was used to determine the optimal concentration of H2O2 and Cys C to stimulate H9c2 cells. We determined the effects of Cys C on oxidative stress and apoptosis levels in H9c2 cells by measuring the activity of dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA), and the expression of apoptosis-related molecules (caspase3/8/9, Bax and Bcl-2). Changes in the activity of the NF-κB signaling pathway in H9c2 cells were also detected. In addition, we made rat MIRI models by ligating the coronary arteries and used Cys C to treat rats to verify the effect of Cys C on MIRI. RESULTS: According to the results of the CCK8 assay, 1000 µM of H2O2 and 15 µM of Cys C were used to stimulate H9c2 cells. Cys C alleviated H2O2-induced H9c2 cell injury, manifested as a decrease in LDH and MDA activity and an increase in SOD activity. Cys C also reduced the apoptosis level in H9c2 cells. The activity of NF-κB signaling pathway in injured H9c2 cells was increased, and stimulation of Cys C could inhibit the NF-κB signaling pathway in H9c2 cells. The application of Cys C in MIRI rats also verified its therapeutic effect on MIRI. CONCLUSIONS: Cys C reduced the oxidative stress and apoptosis levels of cardiomyocytes by inhibiting the activity of NF-κB signaling pathway in cardiomyocytes, thereby reducing cardiomyocyte injury and treating MIRI.

Cistatina C: Biomarcador de riesgo cardiovascular en VIH / Cystatin C: Biomarker of cardiovascular risk in HIV

INTRODUCCIÓN: Los pacientes infectados por el virus de la inmunodeficiencia humana (VIH) tienen mayor riesgo cardiovascular (RCV). El desarrollo de enfermedad cardiovascular (ECV) en esta población involucra factores de RCV tradicionales y factores relacionados con la propia infección, como el estado proinflamatorio crónico, la disfunción inmune y el tratamiento antirretroviral recibido. La cistatina C (CC) ha demostrado utilidad para valorar la presencia de factores de RCV y ECV establecida en población general, ancianos y enfermos renales. Analizamos dicha asociación en una población VIH+. MATERIAL Y MÉTODOS: Estudio analítico, observacional, transversal. Se recogieron factores de RCV y presencia de ECV en pacientes VIH+, obteniendo determinación de CC. Se establecieron 2 grupos: grupo1=CC elevada (≥0,95mg/L) y grupo 2=CC normal (<0,95mg/L). RESULTADOS: Se incluyeron 95 pacientes, grupo1=27 (28,4%) y grupo2=68 (71,5%). Un valor de CC≥0,95mg/L se relacionó con la presencia de ECV (p = 0,01); con aumento de medias de circunferencia de cintura (p = 0,05), circunferencia de cuello (p = 0,04), presión arterial sistólica (p = 0,04), presión arterial diastólica (p = 0,01), puntaje al score de riesgo de Framingham (p = 0,03) y puntaje al score de riesgo de Framingham adaptado para VIH (p = 0,01). Después de realizarse análisis multivariado con incorporación de variables con asociación bivariada a ECV, solo CC≥0,95mg/L continuó relacionándose con ECV. CONCLUSIÓN: CC≥0,95mg/L se relacionó de forma independiente con la presencia de ECV. Este punto de corte también se vinculó a mayores niveles de presión arterial y mayor RCV a 10 años calculado por score de Framingham y score adaptado para población VIH

INTRODUCTION: Patients infected with the human immunodeficiency virus (HIV) have a higher cardiovascular risk (CVR). The development of cardiovascular disease (CVD) in this population involves traditional CVR factors and factors related to the infection itself, such as chronic inflammatory status, immune dysfunction, as well as the antiretroviral therapy received. Cystatin C (CC) has shown to be useful in assessing the presence of CVR factors and CVD established in the general population, the elderly population, and patients with chronic kidney disease. An analysis was performed on this association in an HIV positive population (HIV+). MATERIAL AND METHODS: Analytical, observational, cross-sectional study was conducted, and included collecting information about CVR factors and CVD in HIV+, as well as measuring CC. The patients were divided into 2 groups: Group1=high CC (≥0.95mg/L) and Group2=normal CC (<0.95mg/L). RESULTS: A total of 95 patients were included. Group1=27 (28.4%) and Group2=68 (71.5%). A value of CC≥0.95mg/L was related to the presence of CVD (P=.01). It was also related with and an increase in waist circumference (P=.05), neck circumference (P=.04), systolic blood pressure (P=.04), diastolic blood pressure (P=.01), Framingham score (P=.03), and Framingham score adapted for HIV (P=.01). After performing multivariate analysis with incorporation of variables associated with CVD in the bivariate analysis, only CC≥0.95mg/L continued to be related to CVD. CONCLUSION: CC≥0.95mg/L was independently associated with CVD. This cut-off point was also linked to higher levels of blood pressure, and higher CVR at 10 years using the Framingham Score and Framingham Score adapted for HIV population

Cystatin C improves blood-brain barrier integrity after ischemic brain injury in mice.

Cystatin C, a well-established biomarker of renal function, has been associated with a protective effect against stroke. However, the potential neuroprotective mechanism of cystatin C in ischemic brain injury remains unclear. Our study hypothesized that cystatin C can ameliorate blood-brain barrier (BBB) disruption by up-regulating caveolin-1 expression, thereby improving neurological outcomes in cerebral ischemic injury. Western blotting, immunohistochemistry, immunofluorescence staining, and immunoprecipitation were performed to investigate target proteins. Evans Blue and gelatin zymography were used to examine the effect of cystatin C on BBB disruption. Plasmid and small interfering RNA transfection was used to observe alterations in caveolin-1 and occludin expression induced by changes in cystatin C expression. Intriguingly, our study showed that the expression of both cystatin C and caveolin-1 was increased in middle cerebral artery occlusion-injured mice, and pretreatment with exogenous cystatin C significantly increased caveolin-1 expression, reduced Evans Blue leakage in the injured brain region, and decreased the enzymatic activity of matrix metallopeptidase-9. Meanwhile, our study also showed that the over-expression of cystatin C greatly enhanced caveolin-1 expression, which later increased occludin expression in oxygen-glucose deprivation-exposed brain microvascular endothelial cells. The knockdown of cystatin C induced the opposite outcomes. These experimental results indicate a positive role for cystatin C in the regulation of caveolin-1 and occludin expression in cerebral ischemic injury. Taken together, these data unveil a new mechanism of the regulation of caveolin-1 expression by cystatin C in the maintenance of BBB integrity after ischemic brain injury and provide new clues for the identification of potential therapeutic strategies for stroke.

Cistatina C en el derrame pleural / Cystatin C in Pleural Effusion

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Ausencia de implicación de la cistatina C en el remodelado ventricular y la insuficiencia cardiaca / Absence of cystatin C involvement in ventricular remodelling and heart failure

La cistatina C (CisC) es una proteasa codificada por genes de mantenimiento («housekeeping genes»). Aunque su valor pronóstico en la insuficiencia cardiaca (IC) es bien conocido, se debate si es debido a su mayor precisión en la estimación del filtrado glomerular, o a su implicación en el remodelado ventricular patológico. El propósito de este estudio fue comprobar si la expresión de CisC se modificaba en el miocardio de fetos de diferentes edades y en el de adultos con diversas enfermedades cardiovasculares, así como analizar la correlación entre sus concentraciones séricas y la estructura y morfología cardiaca en un grupo de pacientes con IC. Pacientes y métodos. Se analizaron las correlaciones (test de Pearson y Spearman) entre la CisC sérica y los parámetros ecocardiográficos de 351 pacientes con IC. También se realizó una tinción inmunohistoquímica para CisC, metaloproteinasa 9 (MMP-9) y desmina en 9 muestras de tejido cardiaco procedentes de las autopsias de 4 fetos con diferente edad gestacional y 5 adultos sanos o con enfermedad cardiovascular. Resultados. En pacientes con IC no se encontró correlación entre las concentraciones de CisC y los parámetros cardiacos medidos por ecocardiografía 2D. La inmunohistoquímica mostró una débil tinción de fondo para CisC en todas las muestras, independientemente de la edad y la presencia o no de enfermedades cardiovasculares. Conclusiones. Nuestros resultados sugieren que la CisC no tiene un papel significativo en el remodelado patológico del ventrículo izquierdo en la IC (AU)

Cystatin C (CysC) is a protease encoded by housekeeping genes. Although its prognostic value in heart failure (HF) is well known, it is debatable whether this value is due to the greater accuracy of CysC in calculating the glomerular filtration rate or to its involvement in pathological ventricular remodelling. The aim of this study was to determine whether CysC expression changes in the myocardium of foetuses of different ages and in the myocardium of adults with various cardiovascular diseases, as well as to analyse the correlation between its serum concentrations and cardiac structure and morphology in a patient group with HF. Patients and methods. We analysed the correlations (Pearson's r and Spearman's test) between the serum CysC levels and echocardiographic parameters of 351 patients with HF. We also performed immunohistochemical staining for CysC, metalloproteinase-9 (MMP-9) and desmin in 9 cardiac tissue samples from autopsies of 4 foetuses of different gestational ages and 5 healthy adults or adults with cardiovascular disease. Results. For the patients with HF, there was no correlation between the CysC concentrations and the cardiac parameters measured by 2D echocardiography. The immunohistochemistry showed a weak background staining for CysC in all samples, regardless of age and the presence or absence of cardiovascular diseases. Conclusions. Our results suggest that CysC does not have a significant role in the pathological remodelling of the left ventricle in HF (AU)

Cistatina C: una pieza más de la compleja interacción cardiorrenal en la insuficiencia cardíaca / Cystatin C: one more component of the complex cardiorenal interaction in heart failure

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Cistatina C, muchas respuestas y algunas cuestiones pendientes / Cystatin C, many answers but some unmet questions

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Evaluation of cystatin C activities against HIV.

BACKGROUND & OBJECTIVES: Several host defense proteins known to possess antimicrobial activities are present on mucosal surfaces and are consequently found in body fluids of vertebrates. Naturally occurring protease inhibitors like cystatins, especially cystatin C (cys C), are abundantly present in human seminal plasma. Although its antiviral activity against herpes simplex virus (HSV) has been demonstrated, the role of this protein against HIV is not well studied. Therefore, the aim of the present study was to evaluate the anti-HIV activities of cys C, which is present innately in the male reproductive tract. METHODS: Protein-protein interaction of cys C with various HIV proteins was studied using a commercially available HIV blot and specific interaction with HIV protease was studied by dot-blot technique using commercially available cys C. To purify biologically active cys C from human seminal plasma to be used for subsequent experiments, gel-permeation chromatography followed by affinity chromatography was used. The HIV infectivity inhibition activity of the purified cystatin C was tested in TZM-bl cells. To study its activity on HIV protease, time-course enzyme kinetics studies were performed using spectrometric assay. RESULTS: Cystatin C reacted with some HIV proteins including HIV protease. Biologically active cys C was purified using gel permeation chromatography followed by affinity chromatography. When tested in TZM-bl cells, purified cystatin C demonstrated HIV-infectivity inhibitory activity (IC 50: 0.28 µM). Enzyme kinetic studies demonstrated that it abrogated the action of HIV protease on its substrate. INTERPRETATION & CONCLUSIONS: The present data demonstrate that cystatin C possesses anti-HIV activities. Molecular models need to be designed with this protein which would assist towards prevention/ therapeutics against HIV.

Cystatin C is an Independent Risk Factor and Therapeutic Target for Acute Ischemic Stroke.

Cystatin C (CysC) has been used as a renal function indicator and a strong predictor of cardiovascular events. In this study, we determined the prognostic value of serum CysC in patients with acute ischemic stroke (AIS) and examined its protective role on ischemic brain injury. First-ever stroke patients (601 cases) and control subjects (325 cases) were recruited. Serum CysC level in patients with AIS were significantly higher than that in controls. Multivariate logistic regression analyses showed that elevated CysC is an independent risk factor of AIS; the odds ratio (95 % confidence interval) was 9.80 (3.12-30.83). The integrated population was divided into quintiles according to serum CysC. Compared with the first quintile, the odds ratios of risk for AIS in fourth quintile and fifth quintile were 1.92 (1.08-3.40) and 2.88 (1.49-5.54), respectively. Serum CysC was not associated with neurological deficits and the location of ischemic area; however, serum CysC in patients decreased after one-week therapy. This was consistent with CysC expression after ischemia/reperfusion injury in a mouse focal ischemia/reperfusion injury model. Exogenous CysC exerted neuroprotective effects by reducing infarct volume in this animal stroke model. Therefore, serum CysC is highly associated with AIS and is an independent prediction marker for AIS. Since our findings demonstrated the protection of exogenous CysC on ischemic brain injury, CysC is a novel and promising therapeutic target for AIS.

Induction of autophagy by cystatin C: a potential mechanism for prevention of cerebral vasospasm after experimental subarachnoid hemorrhage.

BACKGROUND: Studies have demonstrated that autophagy pathways are activated in the brain after experimental subarachnoid hemorrhage (SAH) and this may play a protective role in early brain injury. However, the contribution of autophagy in the pathogenesis of cerebral vasospasm (CVS) following SAH, and whether up-regulated autophagy may contribute to aggravate or release CVS, remain unknown. Cystatin C (CysC) is a cysteine protease inhibitor that induces autophagy under conditions of neuronal challenge. This study investigated the expression of autophagy proteins in the walls of basilar arteries (BA), and the effects of CysC on CVS and autophagy pathways following experimental SAH in rats. METHODS: All SAH animals were subjected to injection of 0.3 mL fresh arterial, non-heparinized blood into the cisterna magna. Fifty rats were assigned randomly to five groups: control group (n = 10), SAH group (n = 10), SAH + vehicle group (n = 10), SAH + low dose of CysC group (n = 10), and SAH + high dose of CysC group (n = 10). We measured proteins by western blot analysis, CVS by H&E staining method, morphological changes by electron microscopy, and recorded neuro-behavior scores. RESULTS: Microtubule-associated protein light chain-3, an autophagosome biomarker, and beclin-1, a Bcl-2-interacting protein required for autophagy, were significantly increased in the BA wall 48 h after SAH. In the CysC-handled group, the degree of CVS, measured as the inner BA perimeter and BA wall thickness, was significantly ameliorated in comparison with vehicle-treated SAH rats. This effect paralleled the intensity of autophagy in the BA wall induced by CysC. CONCLUSIONS: These results suggest that the autophagy pathway is activated in the BA wall after SAH and CysC-induced autophagy may play a beneficial role in preventing SAH-induced CVS.

Cystatin C uptake in the eye.

BACKGROUND: As a secreted protein, cystatin C is assumed to play its role in the extracellular compartment, where it can inhibit virtually all cysteine proteases of families C1 (cathepsin B, L, S) and C13 (mammalian legumain-related proteases). Since many of its potential target enzymes in the eye reside in intracellular compartments, we sought evidence for a cellular uptake of the inhibitor in ocular tissues. METHODS: Fluorescence-labeled human cystatin C was injected intravitreally into normal rat eyes. Ocular tissues were subsequently examined using ELISA, fluorescence microscopy, and immunohistochemistry. Cystatin C uptake was additionally studied in an in vitro retina model. RESULTS: Cystatin C administered intravitreally in vivo is taken up into cells of the corneal endothelium and epithelium, the epithelial cells lining the ciliary processes, and into cells in the neuroretina (mostly ganglion cells) and the retinal pigment epithelium. The uptake is demonstrable also in vitro and was, in the neuroretina, found to be a high-affinity system, inhibited by cooling the specimens or by adding the microfilament polymerization inhibitor, cytochalasin D, to the medium. CONCLUSIONS: There is an active, temperature-dependent uptake system for cystatin C into several cell types in the cornea, ciliary body, and retina. The cell types that take up cystatin C are generally the same that contain endogenous cystatin C, suggesting that much or all cystatin C seen intracellularly in the normal eye may have been taken up from the surrounding extracellular space. The uptake indicates that the inhibitor may exert biological functions in intracellular compartments. It is also possible that this uptake system may regulate the extracellular levels of cystatin C in the eye.