Non-specific Alkaline Phosphatase Activity Research Articles

Effects of replacing soybean meal with cottonseed meal on growth, feed utilization and non-specific immune enzyme activities for juvenile white shrimp, Litopenaeus vannamei

Cottonseed meal (CSM) was evaluated as a protein source for partial replacement of soybean meal (SBM) with iron supplementation in diets of juvenile Litopenaeus vannamei. In the control diet (D0), SBM protein was replaced with increasing dietary levels of CSM (0 %, 9.2 %, 15.2 %, 21.3 %, 27.4 %, and 30.4 %) at 0 %, 30 %, 50 %, 70 %, 90 %, and 100 % levels (D0 to D100, respectively) with ferrous sulfate supplementation at level of 0 %, 0.04 %, 0.08 %, 0.1 % and 0.12 %, respectively. An eight-week feeding trial was conducted on shrimp (0.383 ± 0.000 g). Up to 90 % of SBM could be replaced with CSM without causing a significant decrease (P < 0.05) in growth and feed utilization in L. vannamei. Related whole-body composition indexes (moisture, crude protein, crude lipid, ash) were significantly affected with the CSM level in the diets increased (P < 0.05). Regarding serum non-specific immune enzymes, phenoloxidase, acid phosphatase and alkaline phosphatase activities of shrimp were significantly decreased (P < 0.05) with the CSM level in the diets increased. Glutathione peroxidase activity was the highest in shrimp fed the D50 diet, and significantly higher than D0 group (P < 0.05). Total-superoxide dismutase of shrimp fed D90 and D100 diets were significantly lower than that of shrimp fed other diets, whereas malondialdehyde content was significantly higher in the D90 and D100 diets than in the other dietary treatments. Based on the findings in changes in growth and feed utilization, this study found that the optimum ratio is to replaced 87.44 % of SBM with 26.82 % CSM in L. vannamei feed.

Clinical application experience of asfotase alfa for a young patient with childhood hypophosphatasia

Hypophosphatasia (HPP) is a rare hereditary metabolic disease characterized by defective bone and dental mineralization, systemic complications that lead to disability of patients. HPP is classified into six forms according to the age of onset and severity of its clinical picture. The disease is caused by a reduced activity of the tissue nonspecific alkaline phosphatase (TNSALP) and elevated concentrations of pyrophosphates. Asfotase alfa is the only pathogenetic enzyme replacement therapy with recombinant human bone-targeted TNSALP approved for treatment of patients with perinatal, infantile and juvenile‐onset HPP. This treatment is associated with improved skeletal mineralization, respiratory function and overall survival in infants and young children with life-threatening hypophosphatasia. The world experience in application of recombinant alkaline phosphatase in adults is very limited. We present a clinical case that describes the first Russian experience in the use of asfotase alfa in an 18-year-old patient with late diagnosis of childhood-onset HPP.

Identification of common antigens of three pathogenic Nocardia species and development of DNA vaccine against fish nocardiosis

Fish nocardiosis is a chronic granulomatous bacterial disease and three pathogens have been reported so far, including Nocardia asteroids, N. seriolae and N. salmonicida. However, the absence of antigen markers is a bottleneck for developing effective vaccines against fish nocardiosis. In this study, the antigenicity of whole-cell protein of these three pathogenic Nocardia species were profiled by immunoproteomic analysis and 7 common immunogenic proteins were identified as follows: molecular chaperone DnaK (DnaK), molecular chaperone GroEL (GroEL), 30 S ribosomal protein S1 (RpsA), TerD family protein (TerD), FHA domain-containing protein (FHA), 50 S ribosomal protein L7/L12 (RplL) and PspA/IM30 family protein (PspA). Furthermore, the DNA vaccine encoding FHA gene against fish nocardiosis was developed and its efficacy was investigated in hybrid snakehead. The results suggested that it needed at least 7 d to transport pcDNA-FHA DNA vaccine from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection. In addition, non-specific immunity paraments (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα) were used to evaluate the immune response induced in pcDNA-FHA vaccinated hybrid snakehead, it proved that all these mentioned immune activities were significantly enhanced after immunization. The results also showed hybrid snakehead vaccinated with pcDNA-FHA had higher survival rate (79.33%) compared with the controls after challenge with N. seriolae, indicating that the pcDNA-FHA DNA vaccine can supply immune protection against N. seriolae infection. Taken together, this study may warrant further development of these common immunogenic proteins as the antigens for vaccine or diagnosis and facilitate the prevention and treatment of fish nocardiosis.

Comparison of protective efficacy between two DNA vaccines encoding DnaK and GroEL against fish nocardiosis

Fish nocardiosis is a chronic granulomatous bacterial disease mainly caused by three pathogenic bacteria, including Nocardia seriolae, N. asteroids and N. salmonicida. Molecular chaperone DnaK and GroEL were identified to be the common antigens of the three pathogenic Nocardia species in our previous studies. To evaluate the immune protective effect of two DNA vaccines encoding DnaK or GroEL against fish nocardiosis, hybrid snakehead were vaccinated and the immune responses induced by these two vaccines were comparatively analyzed. The results suggested it needed at least 7 d to transport DnaK or GroEL gene from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection after immunization by DNA vaccines. Additionally, non-specific immunity parameters (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα) were used to evaluate the immune responses induced in vaccinated hybrid snakehead. It proved that all the above-mentioned immune activities were significantly enhanced after immunization with these two DNA vaccines. The protective efficacy of pcDNA-DnaK and pcDNA-GroEL DNA vaccines, in terms of relative percentage survival (RPS), were 53.01% and 80.71% respectively. It demonstrated that these two DNA vaccines could increase the survival rate of hybrid snakehead against fish nocardiosis, albeit with variations in immunoprotective effects. Taken together, these results indicated that both pcDNA-DnaK and pcDNA-GroEL DNA vaccines could boost the innate, humoral and cellular immune response in hybrid snakehead and show highly protective efficacy against fish nocardiosis, suggesting that DnaK and GroEL were promising vaccine candidates. These findings will promote the development of DNA vaccines against fish nocardiosis in aquaculture.

Chronic recurrent multifocal osteomyelitis in children with hypophosphatasia explained by anti-inflammatory nucleotidase activity of tissue nonspecific alkaline phosphatase in mesenchymal and hematopoietic cells

Chronic recurrent multifocal osteomyelitis in children with hypophosphatasia explained by anti-inflammatory nucleotidase activity of tissue nonspecific alkaline phosphatase in mesenchymal and hematopoietic cells

Tissue non-specific alkaline phosphatase activity and mineralization capacity of bi-allelic mutations from severe perinatal and asymptomatic hypophosphatasia phenotypes: Results from an in-vitro mutagenesis model

Tissue non-specific alkaline phosphatase activity and mineralization capacity of bi-allelic mutations from severe perinatal and asymptomatic hypophosphatasia phenotypes: Results from an in-vitro mutagenesis model

Lethal perinatal hypophosphatasia caused by a novel compound heterozygous mutation: a case report

BackgroundHypophosphatasia (HPP) is a rare hereditary disorder characterized by defective bone and tooth mineralization and deficiency of tissue non-specific alkaline phosphatase (TNAP) activity. The clinical presentation of HPP is highly variable, and the prognosis for the infantile form is poor.Case presentationThis study reports a male infant diagnosed with lethal perinatal HPP. His gene analysis showed two heterozygous missense variants c.406C > T (p.R136C) and c.461C > T (p.A154V). The two mutations originated separately from his parents, consistent with autosomal recessive perinatal HPP, and the c.461C > T (p.A154V) was the novel mutation. Three-level structure model provide an explanation of the two mutated alleles correlating with the lethal phenotype of our patient. Results of SIFT, PolyPhen_2, and REVEL showed two mutations were pathogenic.ConclusionsWe demonstrated a case of perinatal lethal HPP caused by two heterozygous mutations, and one of which was novel. This finding will prove relevant for genetic counseling and perinatal gene testing for affected families.

Roles of Phosphate in Skeleton.

Phosphate is essential for skeletal mineralization, and its chronic deficiency leads to rickets and osteomalacia. Skeletal mineralization starts in matrix vesicles (MVs) derived from the plasma membrane of osteoblasts and chondrocytes. MVs contain high activity of tissue non-specific alkaline phosphatase (TNSALP), which hydrolyzes phosphoric esters such as pyrophosphates (PPi) to produce inorganic orthophosphates (Pi). Extracellular Pi in the skeleton is taken up by MVs through type III sodium/phosphate (Na+/Pi) cotransporters and forms hydroxyapatite. In addition to its roles in MV-mediated skeletal mineralization, accumulating evidence has revealed that extracellular Pi evokes signal transduction and regulates cellular function. Pi induces apoptosis of hypertrophic chondrocytes, which is a critical step for endochondral ossification. Extracellular Pi also regulates the expression of various genes including those related to proliferation, differentiation, and mineralization. In vitro cell studies have demonstrated that an elevation in extracellular Pi level leads to the activation of fibroblast growth factor receptor (FGFR), Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular signal-regulated kinase) pathway, where the type III Na+/Pi cotransporter PiT-1 may be involved. Responsiveness of skeletal cells to extracellular Pi suggests their ability to sense and adapt to an alteration in Pi availability in their environment. Involvement of FGFR in the Pi-evoked signal transduction is interesting because enhanced FGFR signaling in osteoblasts/osteocytes might be responsible for the overproduction of FGF23, a key molecule in phosphate homeostasis, in a mouse model for human X-linked hypophosphatemic rickets (XLH). Impaired Pi sensing may be a pathogenesis of XLH, which needs to be clarified in future.

Moringa oleifera Lam. prevents the development of high fructose diet-induced fatty liver

Ingestion of fructose-rich diets has been associated with the increase in the global prevalence of obesity, dyslipidemia and non-alcoholic fatty liver disease across all age groups. Phytochemicals from the plant Moringa oleifera Lam. reportedly therapeutically reduce obesity, correct hyperlipidemia and decrease excess hepatic lipid accumulation in adults. In light of the increasing prevalence of diet-induced metabolic dysfunction in children, this study was designed to investigate the protective effects of a methanolic leaf extract of M. oleifera on fructose-induced obesity and hepatic lipid accumulation in growing female rats. Fifty-one 21-day-old female Sprague Dawley rats were weaned and randomly allocated to six treatment groups. The rats had ad libitum access to commercial standard rat food. In addition, they also had plain drinking water (negative control; C), or a 20% fructose solution (H), or 400 mg.kg−1M. oleifera absolute methanolic leaf extract daily with or without fructose solution (M + H or Mo), or 100 mg.kg−1 fenofibrate daily with or without fructose solution for drinking (F + H or FNF). Growth parameters, circulating metabolites, visceral fat pads, hepatic lipid storage, and biochemical markers of general health were assessed after 10 weeks of the interventions. A significant increase in circulating triglycerides (mmol.l−1) (2.30 ± 0.43 vs 1.70 ± 0.30; P < .01, ANOVA) and hepatic lipid stores (11.00 ± 0.21 vs 9.60 ± 0.21%; P < .05, ANOVA) was observed in fructose-fed rats (H) compared with the negative (C) control. The methanolic leaf extracts of M. oleifera (M + H) prevented the fructose induced elevation in hepatic lipid stores (9.80 ± 0.77 vs 11.00 ± 0.21%; P < .05, ANOVA). In the fructose-fed rats the Moringa leaf extracts (M + H) and fenofibrate (F + H) did not prevent the fructose induced hypertriglyceridemia (P < .05, ANOVA) observed in fructose only fed rats (H). However, rats supplemented with M. oleifera leaf extracts alone (Mo) and fenofibrate alone (FNF) had similar triglyceride concentrations to the negative control (C) (P > .05, ANOVA). Visceral fat pads (g) were significantly lower in the fenofibrate treated group (FNF) compared to those on combined fructose and M. oleifera (M + H) (11.00 ± 3.00 vs 17.00 ± 3.40; P < .01, ANOVA). Fructose intake did not result in a significant elevation (P > .05) in plasma cholesterol, however, rats on fenofibrate alone had lower (P < .05) cholesterol concentrations compared to those on fructose alone and compared to the negative control group. Significantly elevated fasting blood glucose levels, tissue non-specific alkaline phosphatase activity, and hepatomegaly were observed only in fenofibrate treated groups (P ≤ .05, ANOVA). No significant differences were observed in the serum insulin concentration (P > .05, ANOVA), HOMA-IR index (P > .05, ANOVA), plasma creatinine (P > .05, ANOVA), body mass gain (P > .05, ANOVA), linear growth (tibia and femur length, P > .05), body mass index (P > .05, ANOVA) and waist circumference (P > .05, ANOVA) between the different treatment groups. The prophylactic potential of M. oleifera methanolic leaf extract should be considered as an alternative prophylactic natural agent against non-alcoholic fatty liver disease, and thus needs to be further explored in humans.

Metal release and cell biological compatibility of beta-type Ti-40Nb containing indium.

Small indium (In) additions up to 5 wt % to the beta-type Ti-40Nb alloy effectively improve its mechanical biofunctionality. The impact on its biocompatibility is addressed in this work. Comparative electrochemical polarization studies and inductively coupled plasma optical emission spectrometry analyses were conducted in Tris-buffered saline (on the basis of 150 mM NaCl) with pH 7.6 and 2.0 at 310 ± 1 K with Ti-6Al-4V as reference. The metal ion releases from beta-type alloys were generally very low, for example, those of In3+ ions from (Ti-40Nb)-4In specimens were below 6 × 10-7 mmol/cm2 . X-ray photoelectron spectroscopy revealed the passivation mainly by Ti- and Nb-oxides with traces of In-oxides as the dominating surface process. In vitro studies demonstrate a better human bone marrow stromal cells (hBMSC) activity on the beta-type alloys in comparison to CP-Ti (grade 2), which is mainly due to their high Nb content. At 24 h after seeding on (Ti-40Nb)-4In the metabolic activity of hBMSC was 1.5-fold higher and after 11 days, the tissue non-specific alkaline phosphatase activity was 1.8-fold higher relative to values for CP-Ti. Surface treatments, like chemical etching or plasma oxidation, change the surface topography and the thickness and composition of the oxide layers, but they are not effective in further improving the cell response. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1686-1697, 2018.

Identifying early pathogenic events during vascular calcification in uremic rats

Vascular calcification in chronic kidney disease is a very complex process traditionally explained in multifactorial terms. Here we sought to clarify relevance of the diverse agents acting on vascular calcification in uremic rats and distinguish between initiating and complicating factors. After 5/6 nephrectomy, rats were fed a 1.2% phosphorus diet and analyzed at different time points. The earliest changes observed in the aortic wall were noticed 11 weeks after nephrectomy: increased Wnt inhibitor Dkk1 mRNA expression and tissue non-specific alkaline phosphatase (TNAP) expression and activity. First deposits of aortic calcium were observed after 12 weeks in areas of TNAP expression. Increased mRNA expressions of Runx2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN, Klotho, cathepsin S, MMP2, and ENPP1 were also found after TNAP changes. Increased plasma concentrations of activin A and FGF23 were observed already at 11 weeks post-nephrectomy, while plasma PTH and phosphorus only increased after 20 weeks. Plasma pyrophosphate decreased after 20 weeks, but aortic pyrophosphate was not modified, nor was the aortic expression of MGP, Msx2, several carbonic anhydrases, osteoprotegerin, parathyroid hormone receptor-1, annexins II and V, and CD39. Thus, increased TNAP and Dkk1 expression in the aorta precedes initial calcium deposition, and this increase is only preceded by elevations in circulating FGF23 and activin A. The expression of other agents involved in vascular calcification only changes at later stages of chronic kidney disease, in a complex branching pattern that requires further clarification.

Characteristics of minerals in vesicles produced by human osteoblasts hFOB 1.19 and osteosarcoma Saos-2 cells stimulated for mineralization

Bone cells control initial steps of mineralization by producing extracellular matrix (ECM) proteins and releasing vesicles that trigger apatite nucleation. Using transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) we compared the quality of minerals in vesicles produced by two distinct human cell lines: fetal osteoblastic hFOB 1.19 and osteosarcoma Saos-2. Both cell lines, subjected to osteogenic medium with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo the entire osteoblastic differentiation program from proliferation to mineralization, produce the ECM and spontaneously release vesicles. We observed that Saos-2 cells mineralized better than hFOB 1.19, as probed by Alizarin Red-S (AR-S) staining, tissue nonspecific alkaline phosphatase (TNAP) activity and by analyzing the composition of minerals in vesicles. Vesicles released from Saos-2 cells contained and were surrounded by more minerals than vesicles released from hFOB 1.19. In addition, there were more F and Cl substituted apatites in vesicles from hFOB 1.19 than in those from Saos-2 cells as determined by ion ratios. Saos-2 and h-FOB 1.19 cells revealed distinct mineralization profiles, indicating that the process of mineralization may proceed differently in various types of cells. Our findings suggest that TNAP activity is correlated with the relative proportions of mineral-filled vesicles and mineral-surrounded vesicles. The origin of vesicles and their properties predetermine the onset of mineralization at the cellular level.

Atypical femur fractures: a distinctive tract of adult hypophosphatasia.

Hypophosphatasia (HPP) is a congenital, rare and heterogeneous bone disorder, characterized by a deficit of calcified tissue mineralization, leading to skeletal deformities and osteomalacia in adults, rickets in infants and children, and fragility fractures and premature loss of dentition in children and adults. The disease is caused by a reduced or absent expression and activity of the tissue non-specific alkaline phosphatase (TNSALP) enzyme, derived from inactivating mutations of the alkaline phosphatase (ALPL) gene. Six different clinical variants have been reported, defined by the onset age and characterized by different degrees of severity. The adult form of HPP presents a wide range of clinical manifestations, many of which are non-specific, mild, and often overlapping with other metabolic bone diseases. Consequently, many cases of adult HPP are, commonly, undiagnosed or misdiagnosed, and, subsequently, wrongly or non-treated with severe consequences for patients and a very negative impact on their quality of life and life expectancy, as well as with costs due to the administration of wrong therapies and treatments of their side effects. The occurrence of a fragility atypical femur fracture in the adulthood can be suspected as a clinical indication of an undiagnosed adult mild form of HPP; and the presence of at least one of this kind of fracture can help in the diagnosis of adult HPP, together with conventional HPP biochemical signs.

Hypophosphatasia: Review of Bone Mineral Metabolism, Pathophysiology, Clinical Presentation, Diagnosis, and Treatment

Hypophosphatasia (HPP) is a rare, inherited form of rickets or osteomalacia due to reduced activity of tissue non-specific alkaline phosphatase (TNSALP) caused by a loss of function mutation in the TNSALP gene marked by a low serum alkaline phosphatase. The ratio of PPi to Pi is crucial in the mineralization process. This process is regulated by the interaction of three phosphatases present in matrix vesicles: the mineralization promotors TNSALP and phosphatase orphan 1 (PHOSPHO1) and the mineralization inhibitor nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1). HPP is seen worldwide affecting all races. The broad-ranging clinical severity of HPP is correlated to the pattern of inheritance and degree of TNSALP activity, with lower levels of activity seen in the more severe autosomal recessive forms. HPP is marked by compromise of phase 2 hydroxyapatite crystal formation beyond the matrix vesicle resulting in skeletal hypomineralization, spontaneous fractures, tooth loss, and chondrocalcinosis. The major forms in declining order of severity include the following: perinatal, infantile, childhood, and adult HPP in addition to OdontoHPP and benign prenatal HPP. Biochemistry typically reveals a low serum alkaline phosphatase and elevated alkaline substrates such as pyridoxal-5′-phosphate (PLP) and phosphoethanolamine (PEA). Asfotase alfa, a recent novel enzyme replacement therapy for the treatment of HPP, has transformed this once lethal disease to one that is less severe.

Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Gene therapy improves dental manifestations in hypophosphatasia model mice.

Hypophosphatasia is a rare inherited skeletal disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL) encoding TNSALP. Early exfoliation of primary teeth owing to disturbed cementum formation, periodontal ligament weakness and alveolar bone resorption are major complications encountered in oral findings, and discovery of early loss of primary teeth in a dental examination often leads to early diagnosis of hypophosphatasia. Although there are no known fundamental treatments or effective dental approaches to prevent early exfoliation of primary teeth in affected patients, several possible treatments have recently been described, including gene therapy. Gene therapy has also been applied to TNSALP knockout mice (Alpl-/- ), which phenocopy the infantile form of hypophosphatasia, and improved their systemic condition. In the present study, we investigated whether gene therapy improved the dental condition of Alpl-/- mice. Following sublethal irradiation (4 Gy) at the age of 2 d, Alpl-/- mice underwent gene therapy using bone marrow cells transduced with a lentiviral vector expressing a bone-targeted form of TNSALP injected into the jugular vein (n = 3). Wild-type (Alpl+/+ ), heterozygous mice (Alpl+/- ) and Alpl-/- mice were analyzed at 9 d of age (n = 3 of each), while Alpl+/+ mice and treated or untreated Alpl-/- mice were analyzed at 1 mo of age (n = 3 of each), and Alpl+/- mice and Alpl-/- mice with gene therapy were analyzed at 3 mo of age (n = 3 of each). A single mandibular hemi-section obtained at 1 mo of age was analyzed using a small animal computed tomography machine to assess alveolar bone formation. Other mandibular hemi-sections obtained at 9 d, 1 mo and 3 mo of age were subjected to hematoxylin and eosin staining and immunohistochemical analysis of osteopontin, a marker of cementum. Immunohistochemical analysis of osteopontin, a marker of acellular cementum, revealed that Alpl-/- mice displayed impaired formation of cementum and alveolar bone, similar to the human dental phenotype. Cementum formation was clearly present in Alpl-/- mice that underwent gene therapy, but did not recover to the same level as that in wild-type (Alpl+/+ ) mice. Micro-computed tomography examination showed that gene therapy improved alveolar bone mineral density in Alpl-/- mice to a similar level to that in Alpl+/+ mice. Our results suggest that gene therapy can improve the general condition of Alpl-/- mice, and induce significant alveolar bone formation and moderate improvement of cementum formation, which may contribute to inhibition of early spontaneous tooth exfoliation.

A single injection of gain-of-function mutant PCSK9 adeno-associated virus vector induces cardiovascular calcification in mice with no genetic modification

Background and aimsStudying atherosclerotic calcification in vivo requires mouse models with genetic modifications. Previous studies showed that injection of recombinant adeno-associated virus vector (AAV) encoding a gain-of-function mutant PCSK9 into mice promotes atherosclerosis. We aimed to study cardiovascular calcification induced by PCSK9 AAV in C57BL/6J mice. Methods10 week-old C57BL/6J mice received a single injection of AAV encoding mutant mPCSK9 (rAAV8/D377Y-mPCSK9). Ldlr–/– mice served as positive controls. Mice consumed a high-fat, high-cholesterol diet for 15 or 20 weeks. Aortic calcification was assessed by fluorescence reflectance imaging (FRI) of a near-infrared calcium tracer. ResultsSerum levels of PCSK9 (0.14 μg/mL to 20 μg/mL, p < 0.01) and total cholesterol (82 mg/dL to 820 mg/dL, p < 0.01) increased within one week after injection and remained elevated for 20 weeks. Atherosclerotic lesion size was similar between PCSK9 AAV and Ldlr–/– mice. Aortic calcification was 0.01% ± 0.01 in PCSK9 AAV mice and 15.3% ± 6.1 in Ldlr–/– mice at 15 weeks (p < 0.01); by 20 weeks, the PCSK9 AAV mice aortic calcification grew to 12.4% ± 4.9. Tissue non-specific alkaline phosphatase activity was similar in PCSK9 AAV mice and Ldlr–/– mice at 15 and 20 weeks, respectively. As example of the utility of this model in testing modulators of calcification in vivo, PCSK9 AAV injection to sortilin-deficient mice demonstrated reduced aortic calcification by 46.3% (p < 0.05) compared to littermate controls. ConclusionsA single injection of gain-of-function PCSK9 AAV into C57BL/6J mice is a useful tool to study cardiovascular calcification in mice with no genetic manipulation.

Abstract 45: Sortilin Regulates Arterial Calcification in Atherosclerotic Mice and Associates With Cardiovascular Risk in Humans

Background: Arterial calcification associates with cardiovascular morbidity and mortality. Human genome-wide association studies linked the SORT1 gene encoding sortilin and coronary artery calcification. We previously demonstrated in vitro that calcification requires sortilin phosphorylation by Fam20C. We hypothesized that sortilin mediates arterial calcification in vivo and that serum sortilin levels can predict cardiovascular events. Results: We assessed the effects of genetic deletion of sortilin ( Sort1 ) in LDL receptor-deficient mice ( Ldlr -/- ) consuming a high-fat, high-cholesterol diet. Sort1 -/- Ldlr -/- mice exhibited 80% lower aortic calcification as compared to control Ldlr -/- mice in intact arteries (fluorescence reflectance imaging of a NIRF calcium tracer, p&lt;0.01). Sort1 -/- Ldlr -/- mice showed 88% lower extracted aortic calcium (p&lt;0.05). Histological analysis revealed less tissue non-specific alkaline phosphatase activity and arterial microcalcification. Transfer of Sort1 -deficient bone marrow cells to irradiated atherosclerotic mice does not alter aortic calcification, indicating that immune cell-derived sortilin may not contribute to sortilin-mediated arterial calcification. In contrast to arterial calcium, Sort1 -deficiency did not alter bone mineralization assessed by μCT and histomorphometry. In a community-based cohort of men aged ≥50 (n=830), abdominal aortic calcification (AAC) was assessed by dual energy X-ray absorptiometry using Kauppila score. Serum sortilin levels were measured by ELISA. After adjustment for confounders (including cholesterol concentration), sortilin levels correlated positively with severe AAC. The highest sortilin quartile associated with higher odds of severe AAC even in men without ischemic heart disease (OR=2.0, p&lt;0.05). Among 745 men followed up prospectively for 7.8 years, major adverse cerebrovascular events occurred in 76 men. Elevated serum sortilin associated with higher event risk (adjusted for confounders, HR=2.8, p&lt;0.001) even in men without diabetes (HR=3.5, p&lt;0.001). Conclusions: Sortilin is a novel therapeutic target to modulate the formation of arterial calcification and a biomarker of both aortic calcification and cardiovascular risk.

Abstract 647: Induction of Cardiovascular Calcification in Non-transgenic Mice via a Single Injection of Pcsk9 Adeno-associated Viral Vector

Background: Studying atherosclerotic calcification in vivo requires mouse models with genetic deletion of low-density lipoprotein receptor (Ldlr) or apolipoprotein E. A previous study showed a rapid induction of atherosclerosis by proprotein convertase subtilisin/kexin type 9 (PCSK9) in mice. Here, we hypothesize that this method is a useful in vivo tool to study cardiovascular calcification in non-genetically modified C57BL/6 mice. Results: 10 week old C57BL/6 mice received a single tail vein injection of recombinant adeno-associated viral vector (AAV) encoding PCSK9 (rAAV8/D377Y-mPCSK9). Ldlr -/- and saline injected C57BL/6 mice served as controls. Mice consumed a high-fat, high-cholesterol (HF/HC) diet for 15-20 weeks. PCSK9 and total cholesterol serum levels were significantly increased within one week after injection and maintained for 20 weeks (cholesterol: 82 mg/dL to 820 mg/dL, p&lt;0.01; PCSK9: 0.14 μg/ml to 20 μg/ml, p&lt;0.01). Total cholesterol levels remained 20-30% lower than those of of Ldlr -/- mice. Atherosclerotic lesion size was similar between PSCK9 and Ldlr -/- mice. Saline injected mice did not show any lesions. Plaque collagen content was 31.9%±6.6 in PCSK9 mice and 62.9%±16.6 in Ldlr -/- mice at 15 weeks of HF/HC diet (p=0.01). However, by 20 weeks, the PCSK9 mice had 57.9%±18.6 plaque collagen, suggesting a different stage of plaque progression. Fluorescence reflectance imaging of a near infrared calcium tracer in intact arteries detected 0.4%±0.4 aortic calcification in PCSK9 mice and 9.7%±1.6 in Ldlr -/- mice at 15 weeks of HF/HC diet (p=0.01); by 20 weeks, the PCSK9 mice had 5.3%±1.0 aortic calcification. Tissue non-specific alkaline phosphatase activity positive lesion area was 7.9%±4.0 and 8.3%±2.6 in PCSK9 mice and 10.8%±2.5 and 12.7%±1.7in Ldlr -/- mice at 15 and 20 weeks, respectively. Immunofluorescence analysis demonstrated accumulation of CD68 and RUNX2-positive cells in the plaques of PCSK9 mice similar to Ldlr -/- . Conclusion: While injection of recombinant AAV encoding PCSK9 into C57BL/6 mice induces atherosclerotic calcification with slower sclerotic plaque remodeling compared to Ldlr -/- mice, it may serve as a useful tool to study cardiovascular calcification in mice independent of their genetic background.

Allopurinol and oxypurinol promote osteoblast differentiation and increase bone formation

Allopurinol and its active metabolite, oxypurinol are widely used in the treatment of gout and hyperuricemia. They inhibit xanthine oxidase (XO) an enzyme in the purine degradation pathway that converts xanthine to uric acid. This investigation examined the effect of allopurinol and oxypurinol on bone formation, cell number and viability, gene expression and enzyme activity in differentiating and mature, bone-forming osteoblasts. Although mRNA expression remained relatively constant, XO activity decreased over time with mature osteoblasts displaying reduced levels of uric acid (20% decrease). Treatment with allopurinol and oxypurinol (0.1–1µM) reduced XO activity by up to 30%. At these concentrations, allopurinol and oxypurinol increased bone formation by osteoblasts ~4-fold and ~3-fold, respectively. Cell number and viability were unaffected. Both drugs increased tissue non-specific alkaline phosphatase (TNAP) activity up to 65%. Osteocalcin and TNAP mRNA expression was increased, 5-fold and 2-fold, respectively. Expression of NPP1, the enzyme responsible for generating the mineralisation inhibitor, pyrophosphate, was decreased 5-fold. Col1α1 mRNA expression and soluble collagen levels were unchanged. Osteoclast formation and resorptive activity were not affected by treatment with allopurinol or oxypurinol. Our data suggest that inhibition of XO activity promotes osteoblast differentiation, leading to increased bone formation in vitro.