Browsing by Subject "Alkaline phosphatase"

Now showing 1 - 6 of 6
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    Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence
    (MyJove Corporation, 2021-07-05) Warden, Cassandra; Simmons, Alan J.; Pasic, Lejla; Pitzer, Ashley; Davies, Sean S.; Layer, Justin H.; Mernaugh, Raymond L.; Kirabo, Annet; Medicine, School of Medicine
    Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.
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    Enhancement of osteoblast activity on nanostructured NiTi/hydroxyapatite coatings on additive manufactured NiTi metal implants by nanosecond pulsed laser sintering
    (Dove Press, 2018) Deng, Biwei; Bruzzaniti, Angela; Cheng, Gary J.; Biomedical Sciences and Comprehensive Care, School of Dentistry
    Background: The osteoinductive behaviors of nitinol (NiTi)-based metal implants for bone regeneration are largely dependent on their surface composition and topology. Continuous-mode laser sintering often results in complete melting of the materials and aggregation of particles, which lack control of heat transfer, as well as microstructural changes during sintering of the nanocomposite materials. Methods: In the current study, in situ direct laser deposition was used to additively manufacture three-dimensional NiTi structures from Ni and Ti powders. The mechanical property of NiTi has been shown to be similar to bone. Nanosecond pulsed laser sintering process was then utilized to generate a nanoporous composite surface with NiTi alloy and hydroxyapatite (HA) by ultrafast laser heating and cooling of Ni, Ti, and HA nanoparticles mixtures precoated on the 3D NiTi substrates; HA was added in order to improve the biocompatibility of the alloy. We then studied the underlying mechanism in the formation of NiTi/HA nanocomposite, and the synergistic effect of the sintered HA component and the nanoporous topology of the composite coating. In addition, we examined the activity of bone-forming osteoblasts on the NiTi/HA surfaces. For this, osteoblast cell morphology and various biomarkers were examined to evaluate cellular activity and function. Results: We found that the nanoscale porosity delivered by nanosecond pulsed laser sintering and the HA component positively contributed to osteoblast differentiation, as indicated by an increase in the expression of collagen and alkaline phosphatase, both of which are necessary for osteoblast mineralization. In addition, we observed topological complexities which appeared to boost the activity of osteoblasts, including an increase in actin cytoskeletal structures and adhesion structures. Conclusion: These findings demonstrate that the pulsed laser sintering method is an effective tool to generate biocompatible coatings in complex alloy-composite material systems with desired composition and topology. Our findings also provide a better understanding of the osteoinductive behavior of the sintered nanocomposite coatings for use in orthopedic and bone regeneration applications.
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    Human Recombinant Alkaline Phosphatase (Ilofotase Alfa) Protects Against Kidney Ischemia-Reperfusion Injury in Mice and Rats Through Adenosine Receptors
    (Frontiers Media, 2022-07-28) Rosin, Diane L.; Hall, J. Perry; Zheng, Shuqiu; Huang, Liping; Campos-Bilderback, Silvia; Sandoval, Ruben; Bree, Andrea; Beaumont, Kevin; Miller, Emily; Larsen, Jennifer; Hariri, Ghazal; Kaila, Neelu; Encarnacion, Iain M.; Gale, Jeremy D.; van Elsas, Andrea; Molitoris, Bruce A.; Okusa, Mark D.; Medicine, School of Medicine
    Adenosine triphosphate (ATP) released from injured or dying cells is a potent pro-inflammatory "danger" signal. Alkaline phosphatase (AP), an endogenous enzyme that de-phosphorylates extracellular ATP, likely plays an anti-inflammatory role in immune responses. We hypothesized that ilofotase alfa, a human recombinant AP, protects kidneys from ischemia-reperfusion injury (IRI), a model of acute kidney injury (AKI), by metabolizing extracellular ATP to adenosine, which is known to activate adenosine receptors. Ilofotase alfa (iv) with or without ZM241,385 (sc), a selective adenosine A2A receptor (A2AR) antagonist, was administered 1 h before bilateral IRI in WT, A2AR KO (Adora2a-/- ) or CD73-/- mice. In additional studies recombinant alkaline phosphatase was given after IRI. In an AKI-on-chronic kidney disease (CKD) ischemic rat model, ilofotase alfa was given after the three instances of IRI and rats were followed for 56 days. Ilofotase alfa in a dose dependent manner decreased IRI in WT mice, an effect prevented by ZM241,385 and partially prevented in Adora2a-/- mice. Enzymatically inactive ilofotase alfa was not protective. Ilofotase alfa rescued CD73-/- mice, which lack a 5'-ectonucleotidase that dephosphorylates AMP to adenosine; ZM241,385 inhibited that protection. In both rats and mice ilofotase alfa ameliorated IRI when administered after injury, thus providing relevance for therapeutic dosing of ilofotase alfa following established AKI. In an AKI-on-CKD ischemic rat model, ilofotase alfa given after the third instance of IRI reduced injury. These results suggest that ilofotase alfa promotes production of adenosine from liberated ATP in injured kidney tissue, thereby amplifying endogenous mechanisms that can reverse tissue injury, in part through A2AR-and non-A2AR-dependent signaling pathways.
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    Juvenile Paget’s Disease From Heterozygous Mutation of SP7 Encoding Osterix (Specificity Protein 7, Transcription Factor Sp7)
    (Elsevier, 2020-08) Whyte, Michael P.; Campeau, Philippe M.; McAlister, William H.; Roodman, G. David; Kurihara, Nori; Nenninger, Angela; Duan, Shenghui; Gottesman, Gary S.; Bijanki, Vinieth N.; Sedighi, Homer; Veis, Deborah J.; Mumm, Steven; Medicine, School of Medicine
    Juvenile Paget's disease (JPD) became in 1974 the commonly used name for ultra-rare heritable occurrences of rapid bone remodeling throughout of the skeleton that present in infancy or early childhood as fractures and deformity hallmarked biochemically by marked elevation of serum alkaline phosphatase (ALP) activity (hyperphosphatasemia). Untreated, JPD can kill during childhood or young adult life. In 2002, we reported that homozygous deletion of the gene called tumor necrosis factor receptor superfamily, member 11B (TNFRSF11B) encoding osteoprotegerin (OPG) explained JPD in Navajos. Soon after, other bi-allelic loss-of-function TNFRSF11B defects were identified in JPD worldwide. OPG inhibits osteoclastogenesis and osteoclast activity by decoying receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) away from its receptor RANK. Then, in 2014, we reported JPD in a Bolivian girl caused by a heterozygous activating duplication within TNFRSF11A encoding RANK. Herein, we identify mutation of a third gene underlying JPD. An infant girl began atraumatic fracturing of her lower extremity long-bones. Skull deformity and mild hearing loss followed. Our single investigation of the patient, when she was 15 years-of-age, showed generalized osteosclerosis and hyperostosis. DXA revealed a Z-score of +5.1 at her lumbar spine and T-score of +3.3 at her non-dominant wrist. Biochemical studies were consistent with positive mineral balance and several markers of bone turnover were elevated and included striking hyperphosphatasemia. Iliac crest histopathology was consistent with rapid skeletal remodeling. Measles virus transcripts, common in classic Paget's disease of bone, were not detected in circulating mononuclear cells. Then, reportedly, she responded to several months of alendronate therapy with less skeletal pain and correction of hyperphosphatasemia but had been lost to our follow-up. After we detected no defect in TNFRSF11A or B, trio exome sequencing revealed a de novo heterozygous missense mutation (c.926C>G; p.S309W) within SP7 encoding the osteoblast transcription factor osterix (specificity protein 7, transcription factor SP7). Thus, mutation of SP7 represents a third genetic cause of JPD.
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    Osteoblast-Specific Overexpression of Human WNT16 Increases Both Cortical and Trabecular Bone Mass and Structure in Mice
    (Oxford University Press, 2016-02) Alam, Imranul; Alkhouli, Mohammed; Gerard-O'Riley, Rita L.; Wright, Weston B.; Acton, Dena; Gray, Amie K.; Patel, Bhavmik; Reilly, Austin M.; Lim, Kyung-Eun; Robling, Alexander G.; Econs, Michael J.; Department of Medicine, IU School of Medicine
    Previous genome-wide association studies have identified common variants in genes associated with bone mineral density (BMD) and risk of fracture. Recently, we identified single nucleotide polymorphisms (SNPs) in Wingless-type mouse mammary tumor virus integration site (WNT)16 that were associated with peak BMD in premenopausal women. To further identify the role of Wnt16 in bone mass regulation, we created transgenic (TG) mice overexpressing human WNT16 in osteoblasts. We compared bone phenotypes, serum biochemistry, gene expression, and dynamic bone histomorphometry between TG and wild-type (WT) mice. Compared with WT mice, WNT16-TG mice exhibited significantly higher whole-body areal BMD and bone mineral content (BMC) at 6 and 12 weeks of age in both male and female. Microcomputer tomography analysis of trabecular bone at distal femur revealed 3-fold (male) and 14-fold (female) higher bone volume/tissue volume (BV/TV), and significantly higher trabecular number and trabecular thickness but lower trabecular separation in TG mice compared with WT littermates in both sexes. The cortical bone at femur midshaft also displayed significantly greater bone area/total area and cortical thickness in the TG mice in both sexes. Serum biochemistry analysis showed that male TG mice had higher serum alkaline phosphatase, osteocalcin, osteoprotegerin (OPG), OPG to receptor activator of NF-kB ligand (tumor necrosis family ligand superfamily, number 11; RANKL) ratio as compared with WT mice. Also, lower carboxy-terminal collagen cross-link (CTX) to tartrate-resistant acid phosphatase 5, isoform b (TRAPc5b) ratio was observed in TG mice compared with WT littermates in both male and female. Histomorphometry data demonstrated that both male and female TG mice had significantly higher cortical and trabecular mineralizing surface/bone surface and bone formation rate compared with sex-matched WT mice. Gene expression analysis demonstrated higher expression of Alp, OC, Opg, and Opg to Rankl ratio in bone tissue in the TG mice compared with WT littermates. Our data indicate that WNT16 is critical for positive regulation of both cortical and trabecular bone mass and structure and that this molecule might be targeted for therapeutic interventions to treat osteoporosis.
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    Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms
    (Wiley, 2016-06) Eleniste, Pierre P.; Patel, Vruti; Posritong, Sumana; Zero, Odette; Largura, Heather; Cheng, Ying-Hua; Himes, Evan R.; Hamilton, Matthew; Baughman, Jenna; Kacena, Melissa A.; Bruzzaniti, Angela; Department of Biomedical and Applied Sciences, School of Dentistry
    Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.