Tissue-nonspecific Alkaline Phosphatase Activity Research Articles

Progesterone regulates tissue non-specific alkaline phosphatase (TNSALP) expression and activity in ovine utero-placental tissues

BackgroundTissue non-specific alkaline phosphatase (TNSALP; encoded by the ALPL gene) has a critical role in the postnatal regulation of phosphate homeostasis, yet how TNSALP activity and expression are regulated during pregnancy remain largely unknown. This study tested the hypothesis that progesterone (P4) and/or interferon tau (IFNT) regulate TNSALP activity during pregnancy in sheep.MethodsIn Exp. 1, ewes were bred and received daily intramuscular injections of either corn oil vehicle (CO) or 25 mg progesterone in CO (P4) for the first 8 days of pregnancy and were hysterectomized on either Day 9, 12, or 125 of gestation. In Exp. 2, ewes were fitted with intrauterine catheters on Day 7 of the estrous cycle and received daily intramuscular injections of 50 mg P4 in CO and/or 75 mg progesterone receptor antagonist (RU486) in CO from Days 8 to 15, and twice daily intrauterine injections of either control proteins (CX) or IFNT (25 µg/uterine horn/d) from Days 11 to 15 (treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT) and were hysterectomized on Day 16.ResultsIn Exp. 1, endometria from ewes administered P4 had greater expression of ALPL mRNA than ewes administered CO on Day 12. TNSALP activity appeared greater in the epithelia, stratum compactum stroma, and endothelium of the blood vessels in the endometrium and myometrium from ewes administered P4 than ewes administered CO on Day 12. On Day 125, TNSALP activity localized to uterine epithelial and endothelial cells, independent of P4 treatment. TNSALP activity in placentomes appeared greater in P4 treated ewes and was detected in endothelial cells and caruncular tissue in P4 treated but not CO treated ewes. In Exp. 2, endometrial homogenates from ewes administered RU486 + P4 + CX had lower TNSALP activity those for P4 + CX and P4 + IFNT ewes. Immunoreactive TNSALP protein appeared greater in the mid- and deep-glandular epithelia in RU486 + P4 + CX treated ewes as compared to the other treatment groups. Enzymatic activity appeared greater on the apical surface of the deep glandular epithelia in endometria from ewes treated with RU486 + P4 + CX compared to the other treatment groups.ConclusionsThese results suggest that P4, but not IFNT, regulates the expression and activity of TNSALP in utero-placental tissues and has the potential to contribute to the regulation of phosphate availability that is critical for conceptus development during pregnancy.

Caveolin-dependent endocytosis regulates arterial calcification

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SFB219 Introduction Patients with chronic kidney disease (CKD) have a significantly elevated cardiovascular risk, which is primarily due to premature vascular aging with arterial calcification - a substantial public health burden. Calcifying extracellular vesicles (EVs) are required for the onset and progression of calcification. Endocytosis and exocytosis are important for EV genesis and cargo loading, thus controlling EV release and propensities. Purpose We aim to investigate endocytosis, endosomal ripening, and exocytosis as critical mechanisms of EV-dependent mineralization. Methods and Results First, we characterized EVs from calcifying vascular smooth muscle cells (SMCs) compared to control SMCs. Western blot analysis revealed an increase in tissue nonspecific alkaline phosphatase (TNAP) among calcifying EVs but not in flotillin abundance. When compared to control EVs, calcifying EVs showed higher TNAP activity. Flow cytometry and a turbidity assay were then used to evaluate EV mineral cargo and aggregation potential. Compared to the control, EVs produced from SMCs cultivated under calcifying conditions had 5-fold more mineral-positive EVs (p=0.004) and a 3-fold higher nucleation potential (p<0.001). Next, endocytosis was blocked by targeting Dynamin-1 and –2, which inhibited caveolin- and clathrin-dependent endocytosis (Dynasore hydrate), or targeting clathrin-terminal domain, which inhibited solely clathrin-dependent endocytosis (Pitstop 2). Dynasore reduced matrix mineralization dose-dependently and lowered mineral-positive EVs by 18%, whereas Pitstop 2 had no effect, suggesting a role of caveolin-dependent endocytosis in EV-mediated calcification. Indeed, Cav-1 silencing inhibited matrix mineralization and Cav-1 protein was more abundant in calcifying EVs than in control EVs. Dynasore and Pitstop-2 did not affect the EV number measured by nanoparticle tracking analysis. Inhibition of endosomal ripening by Rab7 inhibition did not affect SMC calcification. Exocytosis was inhibited by Rab27a inhibition, which reduced mineral deposition (-32%, p=0.017) without affecting EV mineral or EV quantity. Conclusion EVs released from calcifying SMCs showed a higher abundance of mineral and TNAP proteins, suggesting a role in mineral transport and deposition. Our data suggest that caveolin-dependent endocytosis is essential in EV-mediated SMC calcification. Mineral cargo in EVs is independent of Rab7 and Rab27a pathways.

Improvement in quality of life after Asfotase Alfa treatment in adults with Pediatric-onset Hypophosphatasia: data from 5 patient-reported outcome measures

Abstract Hypophosphatasia (HPP) is a rare inherited metabolic disorder caused by deficient tissue-nonspecific alkaline phosphatase activity. This study assessed the impact of treatment with asfotase alfa on patient-reported outcomes (PROs) in adults with pediatric-onset HPP. A longitudinal telephone-based survey was administered to eligible individuals enrolled in a patient support program. Interviews were conducted at study entry (prior to asfotase alfa initiation) and after 3, 6, and 12 months. PROs—Patient Health Questionnaire-9 [PHQ-9], Work Productivity and Activity Impairment–Specific Health Problem [WPAI:SHP], Patient-Reported Outcomes Measurement Information System 29 [PROMIS-29], and Routine Assessment of Patient Index Data 3 [RAPID3]—were assessed at each time point. Appropriate statistical tests were performed to assess score changes. Among 50 enrolled patients (mean age: 46 years [SD: 15.4]; 80% female; 94% white), 49 were evaluable at 3 months, 44 at 6 months, and 29 at 12 months. By Month 3, statistically significant improvements from baseline were detected in PHQ-9 scores (10.6 vs. 5.8 [p < 0.0001]), PROMIS-29 domain scores (overall physical function: 38.0 vs. 43.0 [p = 0.001]; anxiety: 57.5 vs. 51.5 [p = 0.0011]; fatigue: 63.3 vs. 55.3 [p < 0.0001]; sleep disturbances: 58.8 vs. 54.3 [p = 0.0099]; ability to participate in social roles and activities: 42.6 vs. 47.7 [p = 0.0012]; and pain interference: 63.8 vs. 58.4 [p = 0.001]) and RAPID3 domain scores (functional status: 2.7 vs. 1.1 [p < 0.0001]; pain tolerance: 6.0 vs. 3.2 [p < 0.0001]; and global health estimate: 5.1 vs. 2.7 [p < 0.0001]). Improvements persisted at Month 12. Patients also showed improvements in WPAI:SHP domain scores at Month 6 (presenteeism: 39.6% vs. 14.1% [p < 0.0001] and work productivity loss: 41.9% vs. 14.1% [p < 0.0001]). Treatment with asfotase alfa was associated with improved quality of life across several domains.

216 Loss of Endothelial Tissue-Nonspecific Alkaline Phosphatase Exacerbates Microhemorrhages and Neuroinflammation in a Mouse Model of Ischemic Stroke

INTRODUCTION: Despite significant progress in treatment strategies, stroke remains a leading cause of mortality and morbidity. Blood-brain barrier dysfunction is coupled to neuroinflammation and poor outcomes post-stroke. Tissue-nonspecific alkaline phosphatase (TNAP) is a glycophosphatidylinositol-linked protein localized to brain microvascular endothelial cells (BMECs). However, TNAP’s role in BMECs remains unclear. Recent in vitro data from our laboratory demonstrates that conditional loss of BMEC TNAP significantly worsened barrier integrity. Importantly, we have shown that TNAP activity is significantly reduced on cerebral microvessels in the penumbra of wild-type stroke mice. METHODS: To test this, we generated mice with a conditional deletion of TNAP in endothelial cells (VE-cKO) and compared the results to controls (Alplfl/fl). Six-month-old mice underwent photothrombotic ischemic stroke (PTS) or sham surgery. RESULTS: Laser speckle flowmetry confirmed that PTS mice exhibited a sustained loss of cerebral blood flow compared to sham mice (p < 0.0001). No significant differences in cerebral blood flow and infarct size were noted between VE-cKO PTS and Alplfl/fl PTS mice. Seven days post-PTS, mice were euthanized, and brains were evaluated for BBB dysfunction and neuroinflammation. Preliminary evidence demonstrates a significant increase (p < 0.0001) in GFAP-positive astrogliosis in the cortical penumbra of VE-cKO PTS compared to Alplfl/fl PTS mice. Astrogliosis was also coupled to a significant increase in the number of microhemorrhages (p < 0.0001) in VE-cKO PTS compared to Alplfl/fl PTS mice. Interestingly, large molecule BBB permeability evaluation with immunoglobulin G (150 kDa) showed no significant differences between VE-cKO PTS and Alplfl/fl PTS mice. CONCLUSIONS: Ongoing studies will quantitatively assess the extent of BBB permeability utilizing fluorescent tracers of varying sizes. Collectively, these results support a novel mechanism through which loss of TNAP activity is linked to brain microvascular dysfunction post-stroke.

Effectiveness of asfotase alfa for treatment of adults with hypophosphatasia: results from a global registry

BackgroundHypophosphatasia (HPP) is a rare inherited disease caused by deficient activity of tissue-nonspecific alkaline phosphatase. Many adults with HPP have a high burden of disease, experiencing chronic pain, fatigue, limited mobility, and dental issues, contributing to decreased health-related quality of life (HRQoL). HPP may be treated with the enzyme replacement therapy asfotase alfa though real-world data in adults are limited. This analysis was conducted to assess the clinical effectiveness of asfotase alfa among adults in the Global HPP Registry.MethodsThe Global HPP Registry is an observational, prospective, multinational study. Adults ≥ 18 years of age were included in this analysis if they had serum alkaline phosphatase (ALP) activity below the age- and sex-adjusted reference ranges, and/or ALPL variant(s), and received asfotase alfa for ≥ 6 months. Mobility was assessed with the 6-Minute Walk Test (6MWT), and patient-reported outcomes tools were used to assess pain (Brief Pain Inventory-Short Form), quality of life (36-item Short Form Health Survey, version 2 [SF-36v2]), and disability (Health Assessment Questionnaire-Disability Index) at multiple time points from baseline through Month 36. Data were collected as per usual standard of care; patients may not have contributed data at all time points.ResultsA total of 190 patients met the inclusion criteria. For patients with ≥ 1 follow-up measurement, the mean distance achieved on 6MWT increased from 404 m (range 60–632 m) at baseline (n = 31) to 484 m at Month 12 (range 240–739 m; n = 18) and remained above baseline through Month 36 (n = 7). Improvements in mean self-reported pain severity scores ranged from − 0.72 (95% CI: − 1.23, − 0.21; n = 38) to − 1.13 (95% CI: − 1.76, − 0.51; n = 26) and were observed at all time points. Improvements in the Physical Component Summary score of SF-36v2 were achieved by Month 6 and sustained throughout follow-up. There was a trend toward improvement in the Mental Component Summary score of SF-36v2 at most time points, with considerable fluctuations from Months 12 (n = 28) through 36 (n = 21). The most frequent adverse events were injection site reactions.ConclusionsAdults with HPP who received asfotase alfa for ≥ 6 months experienced improvements in mobility, physical function, and HRQoL, which were maintained over 3 years of follow-up.Registration: NCT02306720; EUPAS13514.

Systemic effects of hypophosphatasia characterization of two novel variants in the ALPL gene.

Hypophosphatasia (HPP) is an inborn metabolic error caused by mutations in the ALPL gene encoding tissue non-specific alkaline phosphatase (TNSALP) and leading to decreased alkaline phosphatase (ALP) activity. Although the main characteristic of this disease is bone involvement, it presents a great genetic and clinical variability, which makes it a systemic disease. Patients were recruited based on biochemical assessments. Diagnosis was made by measuring serum ALP and pyridoxal 5-phosphate levels and finally by Sanger sequencing of the ALPL gene from peripheral blood mononuclear cells. Characterization of the new variants was performed by transfection of the variants into HEK293T cells, where ALP activity and cellular localization were measured by flow cytometry. The dominant negative effect was analyzed by co-transfection of each variant with the wild-type gene, measuring ALP activity and analyzing cellular localization by flow cytometry. Two previously undescribed variants were found in the ALPL gene: leucine 6 to serine missense mutation (c.17T>C, L6S) affecting the signal peptide and threonine 167 deletion (c.498_500delCAC, T167del) affecting the vicinity of the active site. These mutations lead mainly to non-pathognomonic symptoms of HPP. Structural prediction and modeling tools indicated the affected residues as critical residues with important roles in protein structure and function. In vitro results demonstrated low TNSALP activity and a dominant negative effect in both mutations. The results of the characterization of these variants suggest that the pleiotropic role of TNSALP could be involved in the systemic effects observed in these patients highlighting digestive and autoimmune disorders associated with TNSALP dysfunction. The two new mutations have been classified as pathogenic. At the clinical level, this study suggests that both mutations not only lead to pathognomonic symptoms of the disease, but may also play a role at the systemic level.

The role of endomembrane homeostasis in vascular calcification

Abstract Background Cardiovascular diseases are a leading cause of mortality, with cardiovascular calcification as a prominent predictor and contributor. Microcalcifications in thin fibrous caps of atherosclerotic plaques create biomechanical instability, leading to plaque vulnerability. Recent work indicated that cellular-derived extracellular vesicles (EVs) are pivotal for microcalcification nucleation. EVs can originate from the endolysosomal system, and FYVE-Type Zinc Finger Containing Phosphoinositide Kinase (PIKfyve), a lipid kinase, plays a key role in the endolysosomal maturation. Purpose We hypothesize that alterations in endomembrane homeostasis will modulate EV cargo and, thereby, vascular calcification. Methods and Results In calcified arteries and vascular smooth muscle cells (SMCs), we observed increased PIKfyve expression. Phosphatidylinositol 3-phosphate (PI3P) - the substrate of PIKfyve - was decreased in cellular membranes of calcifying SMCs (-40%) and recovered by Apilimod, a small molecule PIKfyve inhibitor. Apilimod prevented matrix mineralization and collagen network formation in calcifying SMCs (Fig. 1A) accompanied by reduced pro-collagen 1A1 secretion (-90%). Apilimod inhibited tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA, protein and activity level. PIKfyve silencing using siRNA showed similar results. Furthermore, Apilimod caused a dose-dependent increase of EV release assessed by nanoparticle tracking analysis (Fig. 1B). Analysis of the EV cargo demonstrated that Apilimod reduces TNAP protein abundance and activity (-60%) - a characteristic cargo that may determine EV calcification potential. Apilimod-induced EVs exhibited lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Next, we performed transcriptome and kinome analyses to discover the underlying mechanism of PIKfye-mediated SMC calcification. These data revealed a link to adipocyte-like differentiation and SMC phenotype specifying pathways. Validation supported increased expression of adipogenic transcription factors (CEBPA, PPARG),genes of the cholesterol and fatty acid metabolism pathways (FABP3, CD36) and enhanced fatty acid (C12) uptake (Fig. 1A) by Apilimod; while SMC-specific markers (ACTA2, SM22) and key SMC transcription factors (GATA6, MYOCD) were downregulated. Finally, in Ldlr-deficient mice fed a high-fat, high-cholesterol diet for 15 weeks, Apilimod application for 5 weeks increased PI3P levels (1.6-fold) and decreased vascular calcification (Fig. 1C), but did not alter plaque size or collagen content in atherosclerotic plaques. Conclusion Disrupting endolysosomal maturation with Apilimod promotes the release of EVs with reduced calcification potential and induces a phenotypic adaption towards adipocyte-like SMCs, causing reduced SMC calcification. Anti-arteriosclerotic effects of Apilimod in vivo remain to be further investigated.Figure

Гипофосфатазия у взрослых: как не пропустить редкий диагноз?

Hypophosphatasia (HPP) is a rare hereditary disease characterized by reduced activity of tissue-nonspecific alkaline phosphatase (TNSALP) encoded by ALPL gene, defective mineralization of bone (osteomalacia) and various musculoskeletal manifestations, such as bone deformity, teeth loss, recurrent fractures, chronic bone and muscle pain, reduced muscular strength, etc. Deficiency of TNSALP activity leads to the extracellular accumulation of its substrates, including inorganic pyrophosphate that inhibits bone mineralization. Homozygous and compound heterozygous mutations of the ALPL gene are associated with a lower TNSALP activity and HPP severity. However, heterozygous dominant negative variants can also result in clinical manifestations. The authors present two adult patients with HPP and discuss diverse clinical features of the disease and the efficacy of asfotase alpha, the human recombinant enzyme-replacement therapy that replaces deficient TNSALP. In adults and adolescents with pediatric-onset HPP, treatment with asfotase alfa was associated with improved functional abilities and health-related quality of life.

Evidence that pyrophosphate acts as an extracellular signalling molecule to exert direct functional effects in primary cultures of osteoblasts and osteoclasts.

Extracellular pyrophosphate (PPi) is well known for its fundamental role as a physiochemical mineralisation inhibitor. However, information about its direct actions on bone cells remains limited. This study shows that PPi decreased osteoclast formation and resorptive activity by ≤50%. These inhibitory actions were associated with reduced expression of genes involved in osteoclastogenesis (Tnfrsf11a, Dcstamp) and bone resorption (Ctsk, Car2, Acp5). In osteoblasts, PPi present for the entire (0-21days) or latter stages of culture (7-21/14-21days) decreased bone mineralisation by ≤95%. However, PPi present for the differentiation phase only (0-7/0-14days) increased bone formation (≤70%). Prolonged treatment with PPi resulted in earlier matrix deposition and increased soluble collagen levels (≤2.3-fold). Expression of osteoblast (RUNX2, Bglap) and early osteocyte (E11, Dmp1) genes along with mineralisation inhibitors (Spp1, Mgp) was increased by PPi (≤3-fold). PPi levels are regulated by tissue non-specific alkaline phosphatase (TNAP) and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). PPi reduced NPP1 expression in both cell types whereas TNAP expression (≤2.5-fold) and activity (≤35%) were increased in osteoblasts. Breakdown of extracellular ATP by NPP1 represents a key source of PPi. ATP release from osteoclasts and osteoblasts was decreased ≤60% by PPi and by a selective TNAP inhibitor (CAS496014-12-2). Pertussis toxin, which prevents Gαi subunit activation, was used to investigate whether G-protein coupled receptor (GPCR) signalling mediates the effects of PPi. The actions of PPi on bone mineralisation, collagen production, ATP release, gene/protein expression and osteoclast formation were abolished or attenuated by pertussis toxin. Together these findings show that PPi, modulates differentiation, function and gene expression in osteoblasts and osteoclasts. The ability of PPi to alter ATP release and NPP1/TNAP expression and activity indicates that cells can detect PPi levels and respond accordingly. Our data also raise the possibility that some actions of PPi on bone cells could be mediated by a Gαi-linked GPCR.

THU459 Hypophosphatasia With Normal ALPL Gene Test; A Case Report

Abstract Disclosure: K. Alkwatli: None. L.Z. Khan: None. Introduction: Hypophosphatasia is a rare hereditary disorder caused by loss of tissue nonspecific alkaline phosphatase activity, an essential enzyme in phosphate metabolism. Severe cases present perinatally and in early childhood. Mild cases can present in adulthood and can be misdiagnosed. We present an interesting patient who was clinically diagnosed with hypophosphatasia despite negative molecular genetic testing. Case presentation: 40 years-old female with a history of fibromyalgia and multiple bone fractures presented to the clinic with a long-standing history of multiple cavities, severe fatigue, headaches, muscle cramps, and arthralgia. The patient had numerous fractures throughout her life that started at age five without significant trauma, including wrist, forearm, and metatarsal bones. Her family history was negative. Laboratory testing showed low alkaline phosphatase levels on multiple occasions, 28,29,30 U/L (reference range 34-123). Her vitamin B6 was elevated at 309 nmol/L (20-125). Her zinc, calcium, vitamin D, and liver function tests were all within normal limits. Bone density scan showed a Z score of -1.0 at the lumbar spine and a Z score of -0.3 at the left femoral neck. Molecular genetic testing of the ALPL gene was negative. Hypophosphatasia was diagnosed based on her clinical and lab assessments, and she is being treated with Asfotase alfa. Her clinical course is yet to be determined. Discussion: ALPL gene is responsible for encoding the tissue nonspecific alkaline phosphatase (TNALP), the main enzyme affected in patients with hypophosphatasia. TNALP degrades inorganic pyrophosphate (PPi) to inorganic phosphate (Pi), an essential element for bone mineralization. It also facilitates vitamin B6 to cross the cell membranes. Therefore, loss of function mutations of the ALPL gene cause accumulation of PPi, which impairs the calcium-phosphate hemostasis and causes damage to multiple organs. In addition, the accumulation of the active form of vitamin B6 extracellularly can result in neurological damage.Patients present with a broad spectrum of musculoskeletal, renal, dental, and neurological symptoms. The variability of the phenotypic magnitudes is thought to be due to heterogeneity in the ALPL gene mutations. Over 400s of ALPL gene mutations have been identified in patients with hypophosphatasia. Novel variants are increasingly being reported.Negative genetic testing of the ALPL gene can still be seen in patients with hypophosphatasia due to either a variant that is undetectable by current testing methodology or if the causative gene/s are not on the ALPL gene panel. Conclusion: Genetic testing is helpful in confirming the disease and providing genetic counseling; however, treatment should still be considered if patients have sufficient clinical and laboratory findings. Presentation: Thursday, June 15, 2023

OR29-02 Energy Deficient Osteoblasts Inefficiently Mineralize Developing Bone In Sheep With Hypophosphatasia

Abstract Disclosure: J.C. Bertels: None. M. McCord: None. L.G. White: None. S. Huggins: None. D. Landrock: None. E.D. Giles: None. L.J. Suva: None. D. Gaddy: None. Hypophosphatasia (HPP) is a rare genetic disorder characterized by low enzymatic activity of tissue nonspecific alkaline phosphatase (TNSALP), resulting in decreased bone mineralization, muscle weakness, and premature loss of deciduous teeth. All HPP phenotypes have been characterized in our CRISPR/Cas9 gene-edited sheep model of HPP. Previous studies identified small, abnormal mitochondria in HPP sheep cells, leading to the hypothesis that reduced bone mineralization may result from decreased osteoblast (OB) differentiation and cellular energetics. To test this idea, iliac crest bone biopsies and sternal bone marrow stromal cells (BMSCs) were harvested from 1 yr old WT and HPP sheep (n=3 per genotype). Histomorphometric analysis of bone biopsies from HPP sheep revealed increased unmineralized osteoid matrix compared to WT, indicative of decreased OB activity, whereas osteoblast and osteoclast number were unchanged. To determine whether BMSCs from HPP sheep were altered from WT in regard to differentiation capacity toward the adipocyte (AD) or OB lineage, 21 day cultures of BMSCs in OB or AD differentiation medium were performed. HPP BMSCs had significantly increased AD differentiation and similar OB differentiation compared to WT. Next, high resolution respirometry of undifferentiated and differentiating cells toward either the OB or AD lineage using Seahorse XF analysis to measure oxidative respiration and glycolysis on days 0, 7, 14 and 21 of both OB and AD differentiation was performed. Results demonstrated that both undifferentiated and differentiating HPP cells (to OB and AD) had significantly decreased oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), reflective of diminished maximal and ATP-linked oxidative respiration and glycolysis. When measured, intracellular ATP levels in differentiating BMSCs, both undergoing osteoblastogenic and adiopogenic differentiation, showed a higher accumulation of ATP. Collectively, these data provide the first evidence that decreased bone mineralization in HPP sheep results from decreased BMSC and OB energetics through both oxidative respiration and glycolysis. In addition, the data demonstrate that reduced TNSALP activity also leads to enhanced bone marrow adipogenesis. Coupled with our previous results demonstrating reduced oxidative respiration and glycolysis in sheep skeletal muscle, these findings demonstrate a fundamental requirement for TNSALP activity in the maintenance of normal cellular respiration and purinergic metabolism in cells of the musculoskeletal system. Moreover, they provide important insight into the potential for targeting increased cellular respiration to alleviate the deleterious effects of HPP. Presentation: Sunday, June 18, 2023

Dentin Matrix Protein 1 Regulates Mineralization of MC3T3-E1 Cells via the TNAP-ANK-ENPP1 Axis.

Dentin matrix protein 1 (DMP1) is central to matrix mineralization. Clarification of the function of DMP1 is crucial to understanding normal bone formation and pathological calcification. The tissue-nonspecific alkaline phosphatase (TNAP) -progressive ankylosing enzyme (ANK) -extracellular nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) axis induces deposition of hydroxyapatite (HA) and pyrophosphate dehydrate (CPPD) by regulating pyrophosphate (PPi). Here, we investigated the mechanism by which DMP1 and the TNAP-ANK-ENPP1 axis participate in mineralization. Expression of DMP1, TNAP, NPP1, and ANK genes in MC3T3-E1 cells was detected by RT-qPCR before and after treatment with DMP1 siRNA. An enzyme-linked immunosorbent assay was used to determine expression of DMP1 protein, TNAP activity was detected by SIGMAFAST p-nitrophenyl phosphate tablets, and mineralization of osteoblasts was determined by alizarin red staining. PPi levels were determined radiometrically and equalized for cell DNA. Levels of calcium, inorganic phosphate, zinc, and magnesium were assessed by standard laboratory techniques. After DMP1 gene silencing, expressions of TNAP, ENPP1, and ANK were correspondingly reduced. DMP1 altered extravesicular and intravesicular ion levels through the TNAP-ENPP1-ANK axis in MC3T3-E1 cells. DMP1 regulated mineralization of MC3T3-E1 cells via the TNAP-ANK-ENPP1 axis and affected TNAP activity by two processes-rapid regulation of the Zn2+ transporter (ZnT) and transcriptional regulation of hysteresis. However, DMP1 may affect expression of ENPP1 and ANK only via hysteresis transcriptional regulation. DMP1, as a calcium trap or catalytic enzyme, appears to have a role in collagen mineralization.

Hypophosphatasia: from birth to adulthood.

Hypophosphatasia (HPP) is an inherited disease caused by a low activity of tissue-nonspecific alkaline phosphatase, a hydrolase that removes phosphate groups from many molecules. Decreased alkaline phosphatase activity leads to the accumulation of three main metabolites, i.e., pyridoxal 5´-phosphate (PLP), inorganic pyrophosphate (PPi), and phosphoethanolamine. Impairment in PLP dephosphorylation induces seizures, while PPi accumulation inhibits bone mineralization. Clinically, HPP has a wide spectrum of presentations, ranging from neonatal death to an apparent lack of symptoms. This disease is classified into six subtypes according to the age at onset of first signs or symptoms. The clinical manifestations of the disease include rickets-like bone changes, bone demineralization, fragility fractures, reduced muscular strength, chest deformity, pulmonary hypoplasia, nephrolithiasis, nephrocalcinosis, and chondrocalcinosis. Treatment of HPP consists of enzyme replacement therapy. Before this therapy was approved, treatment was palliative and associated with high morbidity and mortality. Asfotase alfa has changed the prognosis of the disease by reducing bone deformity and improving bone mineralization, lung function, and muscle weakness, among other benefits. In adults, teriparatide and anti-sclerostin antibody have been used off-label in selected cases, demonstrating benefit in accelerating fracture healing and in concomitant treatment of osteoporosis. This review summarizes the main aspects of HPP and identifies the particularities of the disease in adult patients.

Disulfide Bonds Are Not Necessary for Intrinsic TNSALP Activity.

Recent developments in single-molecule enzymology (SME) have allowed for the observation of subpopulations present in enzyme ensembles. Tissue-nonspecific alkaline phosphatase (TNSALP), a homodimeric monophosphate esterase central to bone metabolism, has become a model enzyme for SME studies. TNSALP contains two internal disulfide bonds that are critical for its effective dimerization; mutations in its disulfide bonding framework have been reported in patients with hypophosphatasia, a rare disease characterized by impaired bone and tooth mineralization. In this paper, we present the kinetics of these mutants and show that these disulfide bonds are not crucial for TNSALP enzymatic function. This surprising result reveals that the enzyme's active conformation does not rely on its disulfide bonds. We posit that the signs and symptoms seen in hypophosphatasia are likely not primarily due to impaired enzyme function, but rather decreased enzyme expression and trafficking.

Abstract 19: Loss Of Endothelial Tissue-nonspecific Alkaline Phosphatase Modifies Sensorimotor Deficits In Chronic Ischemic Stroke

Tissue nonspecific alkaline phosphatase (TNAP) is an ectoenzyme that is known for its anti-inflammatory properties in the immune system. Although TNAP is present in nearly every cell, it is highly abundant in brain endothelial cells (BEC). However, the role of TNAP in the BECs and other cells in the neurovascular unit is poorly understood. Our laboratory has previously identified a role for BEC TNAP in vascular integrity. While the loss of cortical TNAP activity has been observed in ischemic stroke, the functional outcomes that result from the loss of the enzyme are less clear. The objective of this study is to determine how TNAP affects post-stroke functional outcomes. We hypothesized that the loss of endothelial TNAP worsens post-stroke sensorimotor deficits. To test this, we generated mice with a conditional deletion of Alpl , the gene that codes for TNAP, in endothelial cells (VE-cKO mice) and compared the results to their littermate controls ( Alpl fl/fl ). Male and female six month-old mice (n=40) received a photothrombotic ischemic stroke (PTS) or sham surgery followed by daily neurological assessment (Clark’s test) and comprehensive sensorimotor testing, i.e. corner test, rotarod, open field, and gait analysis (DigiGait), over a seven-day period. Laser speckle flowmetry confirmed that PTS mice exhibited a sustained loss of cerebral blood flow compared to sham mice (p<0.0001). Daily neurological scoring showed higher (worse) scores in VEcKO PTS-mice compared to littermate controls (p=0.004). Quantification of total spontaneous movement via open field testing showed a significant interaction among stroke, genotype, and time (p=0.014). Gait analysis showed that the post-stroke stance/swing ratio, a measure of stability, differed between the left and right hind paws in VEcKO mice compared to controls: right hind paw- genotype x stroke interaction (p=0.041), and left hind paw- genotype x surgery x sex interaction (p=0.005). Significant interactions between surgery and sex were also observed in the right fore paw for stride (p=0.02), stride frequency (p=0.00026), and stride length (p=0.02). These results demonstrate a novel role for endothelial TNAP in functional recovery post-stroke.

Anticalcification effects of DS-1211 in pseudoxanthoma elasticum mouse models and the role of tissue-nonspecific alkaline phosphatase in ABCC6-deficient ectopic calcification

Pseudoxanthoma elasticum (PXE) is a multisystem, genetic, ectopic mineralization disorder with no effective treatment. Inhibition of tissue-nonspecific alkaline phosphatase (TNAP) may prevent ectopic soft tissue calcification by increasing endogenous pyrophosphate (PPi). This study evaluated the anticalcification effects of DS-1211, an orally administered, potent, and highly selective small molecule TNAP inhibitor, in mouse models of PXE. Calcium content in vibrissae was measured in KK/HlJ and ABCC6-/- mice after DS-1211 administration for 13–14 weeks. Pharmacokinetic and pharmacodynamic effects of DS-1211 were evaluated, including plasma alkaline phosphatase (ALP) activity and biomarker changes in PPi and pyridoxal-phosphate (PLP). Anticalcification effects of DS-1211 through TNAP inhibition were further evaluated in ABCC6-/- mice with genetically reduced TNAP activity, ABCC6-/-/TNAP+/+ and ABCC6-/-/TNAP+/-. In KK/HlJ and ABCC6-/- mouse models, DS-1211 inhibited plasma ALP activity in a dose-dependent manner and prevented progression of ectopic calcification compared with vehicle-treated mice. Plasma PPi and PLP increased dose-dependently with DS-1211 in ABCC6-/- mice. Mice with ABCC6-/-/TNAP+/- phenotype had significantly less calcification and higher plasma PPi and PLP than ABCC6-/-/TNAP+/+ mice. TNAP plays an active role in pathomechanistic pathways of dysregulated calcification, demonstrated by reduced ectopic calcification in mice with lower TNAP activity. DS-1211 may be a potential therapeutic drug for PXE.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

Clinical and Genetic Characteristics of Pediatric Patients with Hypophosphatasia in the Russian Population.

(1) Hypophosphatasia (HPP) is a rare inherited disease caused by mutations (pathogenic variants) in the ALPL gene which encodes tissue-nonspecific alkaline phosphatase (TNSALP). HPP is characterized by impaired bone mineral metabolism due to the low enzymatic activity of TNSALP. Knowledge about the structure of the gene and the features and functions of various ALPL gene variants, taking into account population specificity, gives an understanding of the hereditary nature of the disease, and contributes to the diagnosis, prevention, and treatment of the disease. The purpose of the study was to describe the spectrum and analyze the functional features of the ALPL gene variants, considering various HPP subtypes and clinical symptoms in Russian children. (2) From 2014-2021, the study included the blood samples obtained from 1612 patients with reduced alkaline phosphatase activity. The patients underwent an examination with an assessment of their clinical symptoms and biochemical levels of TNSALP. DNA was isolated from dried blood spots (DBSs) or blood from the patients to search for mutations in the exons of the ALPL gene using Sanger sequencing. The PCR products were sequenced using a reagent BigDye Terminator 3.1 kit (Applied Biosystems). Statistical analysis was performed using the GraphPad Prism 8.01 software. (3) The most common clinical symptoms in Russian patients with HPP and two of its variants (n = 22) were bone disorders (75%), hypomyotonia (50%), and respiratory failure (50%). The heterozygous carriage of the causal variants of the ALPL gene was detected in 225 patients. A total of 2 variants were found in 27 patients. In this group (n = 27), we identified 28 unique variants of the ALPL gene, of which 75.0% were missense, 17.9% were frameshift, 3.6% were splicing variants, and 3.6% were duplications. A total of 39.3% (11/28) of the variants were pathogenic, with two variants being probably pathogenic, and 15 variants had unknown clinical significance (VUS). Among the VUS group, 28.6% of the variants (7/28) were discovered by us for the first time. The most common variants were c.571G > A (p.Glu191Lys) and c.1171del (Arg391Valfs*12), with frequencies of 48.2% (13/28) and 11% (3/28), respectively. It was found that the frequency of nonsense variants of the ALPL gene was higher (p < 0.0001) in patients with the perinatal form compared to the infantile and childhood forms of HPP. Additionally, the number of homozygotes in patients with the perinatal form exceeded (p < 0.01) the frequencies of these genotypes in children with infantile and childhood forms of HPP. On the contrary, the frequencies of the compound-heterozygous and heterozygous genotypes were higher (p < 0.01) in patients with infantile childhood HPP than in perinatal HPP. In the perinatal form, residual TNSALP activity was lower (p < 0.0005) in comparison to the infantile and childhood (p < 0.05) forms of HPP. At the same time, patients with the heterozygous and compound-heterozygous genotypes (mainly missense variants) of the ALPL gene had greater residual activity (of the TNSALP protein) regarding those homozygous patients who were carriers of the nonsense variants (deletions and duplications) of the ALPL gene. Residual TNSALP activity was lower (p < 0.0001) in patients with pathogenic variants encoding the amino acids from the active site and the calcium and crown domains in comparison with the nonspecific region of the protein.

Novel Therapeutic Agents for Rare Diseases of Calcium and Phosphate Metabolism.

The last decade has been revolutionary regarding the management of rare bone diseases caused by impaired calcium and phosphate metabolism. Elucidation of the underlying genetic basis and pathophysiologic alterations has been the determinant factor for the development of new, disease-specific treatment agents. The phosphaturic hormone Fibroblast Growth Factor 23 (FGF23) possesses a critical role in the pathogenesis of various hypophosphatemic disorders. Among them, the genetic disorder of X-linked hypophosphatemia and the acquired syndrome of tumor-induced osteomalacia, although very rare, have attracted the scientific community's attention towards designing an FGF23-inhibitor as a potential specific therapy. The monoclonal antibody burosumab was approved for the treatment of children and adult patients with X-linked hypophosphatemia and recently for tumor-induced osteomalacia patients, demonstrating benefits regarding their symptoms, biochemical profile and bone mineralization status. Asfotase alfa is a hydroxyapatite-targeted recombinant alkaline phosphatase, an enzymatic replacement therapy, substituting the defective activity of tissue non-specific alkaline phosphatase, in patients suffering from hypophosphatasia. Promising data regarding its favorable effect on survival rate, bone quality, fracture healing, muscle strength, mobility, respiratory function, and general quality of life have led to the approval of the drug for the treatment of childhood-onset hypophosphatasia. Given the high costs of treatment for both agents and their limited clinical use until now, more data are needed to define patients' characteristics that make them ideal candidates for therapy. Long-term safety issues also need to be clarified.

NPP1 and TNAP hydrolyze ATP synergistically during biomineralization.

Matrix vesicles (MVs) are a special class of extracellular vesicles released by mineralizing cells during bone and tooth mineralization that initiate the precipitation of apatitic minerals by regulating the extracellular ratio between inorganic phosphate (Pi), a calcification promoter, and pyrophosphate (PPi), a calcification inhibitor. The Pi/PPi ratio is thought to be controlled by two ecto-phosphatases present on the outer leaflet of the MVs' membrane: ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) that produces PPi as well as Pi from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PPi to generate Pi. However, if and how these enzymes act in concert in MVs are still unclear. Herein, we investigated the role of NPP1 and TNAP in ATP hydrolysis during MV-mediated biomineralization using proteoliposomes as a biomimetic model for MVs. Proteoliposomes composed by 1,2-dipalmitoylphosphatidylcholine (DPPC) and harboring NPP1 alone, TNAP alone, or both together at different molar ratios (1:1, 10:1, and 1:10) were fabricated. After 48h of incubation with ATP, TNAP-containing proteoliposomes consumed more ATP than NPP1-containing vesicles (270 and 210nmol, respectively). Both types of vesicles comparatively formed ADP (205 and 201nmol, respectively), while NPP1-containing vesicles hydrolyzed AMP less efficiently than TNAP-containing proteoliposomes (10 and 25nmol, respectively). In vitro mineralization assays showed that in the presence of ATP, TNAP-harboring proteoliposomes mineralized through a sigmoidal single-step process, while NPP1-harboring vesicles displayed a two-step mineralization process. ATR-FTIR analyses showed that the minerals produced by TNAP-harboring proteoliposomes were structurally more similar to hydroxyapatite than those produced by NPP1-harboring vesicles. Our results with proteoliposomes indicate that the pyrophosphohydrolase function of NPP1 and the phosphohydrolase activity of TNAP act synergistically to produce a Pi/PPi ratio conducive to mineralization and the synergism is maximal when the two enzymes are present at equimolar concentrations. The significance of these findings for hypophosphatasia is discussed.