Non-specific Alkaline Phosphatase Research Articles

Putting forward novel sulfonamide-thiazole-pyrazoline hybrids as potential central core structure for the development of non-specific b-TNAP and c-IAP inhibitors.

Alkaline phosphatases (ALPs) play crucial role in various functions of human body, such as bone formation, metabolism in liver and intestines, and transfer of nutrients from mother to fetus during pregnancy. However, their overexpression is associated with severe consequences in different patients, such as deposition of minerals in dialysis patients also called coronary calcification and increased bone turnover in patients facing cancer metastization. Due to their involvement in crucial functions of human body and association with such harsh consequences, there is need of newer efficient ALP inhibitors that can tackle ALP excess without derailing the progress of normal functions. In this study, we reported synthesis and biological evaluation of novel series of sulfonamide-thiazole-pyrazoline hybrids (8a-j). The substitutions on the terminal phenyl groups of pyrazoline ring were designed as the basis for the SAR; however, all compounds showed efficient ALP (b-TNAP and c-IAP) inhibition activity, with 8c (IC50=0.87±0.11μM) being the most potent against b-TNAP and 8f (IC50=2.11±0.34μM) being the most potent against c-IAP. In addition, in silico studies were also conducted to provide insights into the binding interactions, drug-likeness and charge density of structures. The IC50 results for all of the compounds were better compared to both references irrespective of the substitutions attached, therefore the sulphonamide-thiazole-pyrazoline hybrid core can be put forward as the central core for designing new drugs for non-specific ALP inhibition.

Diagnostic and New Therapeutic Approaches to Two Challenging Pediatric Metabolic Bone Disorders: Hypophosphatasia and X-linked Hypophosphatemic Rickets.

The diagnosis and management of metabolic bone disease among children can be challenging. This difficulty could be due to many factors, including limited awareness of these rare conditions, the complex pathophysiology of calcium and phosphate homeostasis, the overlapping phenotype with more common disorders (such as rickets), and the lack of specific treatments for these rare disorders. As a result, affected individuals could experience delayed diagnosis or misdiagnosis, leading to improper management. In this review, we describe the challenges facing diagnostic and therapeutic approaches to two metabolic bone disorders (MBD) among children: hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH). We focus on explaining the pathophysiological processes that conceptually underpin novel therapeutic approaches, as well as these conditions' clinical or radiological similarity to nutritional rickets. Particularly in areas with limited sun exposure and among patients not supplementing vitamin D, nutritional rickets are still more common than HPP and XLH, and pediatricians and primary physicians frequently encounter this disorder in their practices. More recently, our understanding of these disorders has significantly improved, leading to the development of novel therapies. Asfotas alfa, a recombinant, human- tissue, nonspecific alkaline phosphatase, improved the survival of patients with HPP. Burosumab, a human monoclonal anti-FGF23 antibody, was recently approved as a specific therapy for XLH. We also highlight the current evidence on these two specific therapies' safety and effectiveness, though long-term data are still needed. Both HPP and XLH are multisystemic disorders that should be managed by multidisciplinary teams. Finally, recognizing these conditions in early stages will enable affected children and young adults to benefit from newly introduced, specific therapies.

7336 Decades In The Dark: The Long Road To Diagnosing And Treating Hypophosphatasia

Abstract Disclosure: V.A. Mendpara: None. G. Madrigal Loria: None. L.Z. Khan: None. Background: Hypophosphatasia (HPP) is a rare bone disorder that results from deficient activity of the tissue non-specific isoenzyme alkaline phosphatase (ALP).[1] This case highlights a patient whose diagnosis of this treatable genetic condition was significantly delayed for almost two decades, despite receiving care at a prominent academic institution. The patient's persistently low ALP levels in routine lab tests were overlooked by multiple clinicians, leading to a delay in her care and eventual treatment. Case presentation: A 61-year-old Caucasian woman presented at the endocrinology clinic with complaints of severe fatigue, generalized pain, multiple childhood fractures, and ongoing dental issues. The rheumatology clinic to which she was referred for polyarthralgia diagnosed her with Hypophosphatasia (HPP) after noting chronically low alkaline phosphatase (ALP) levels from her previous charts ranging from 15 to 24 U/L (normal range: 34 to 123 U/L) over two decades. Her serum calcium, parathyroid hormone (PTH), and 25-hydroxy vitamin D levels were within normal ranges. A DEXA scan revealed normal bone density, effectively ruling out osteoporosis. Further investigation showed elevated Vitamin B6 levels at 219.6 nmol/L (normal range: 20.0 to 125.0 nmol/L), raising suspicion for HPP. The patient's family history provided additional insight, with her mother having a history of short stature and fractures and her son experiencing premature tooth loss at the age of 2. A genetic evaluation identified a heterozygous pathogenic variant in the ALPL gene (c.881A>C, p.Asp294Ala), confirming the diagnosis of autosomal dominant HPP. Initiating treatment with Asfotase alfa, a medication targeting HPP, resulted in a marked improvement in the patient's symptoms. Discussion: This case shows the diagnostic complexities of HPP, stressing the significance of actively screening for this condition via ALP levels in routine laboratory testing or during osteoporosis evaluations. The diagnostic process is critical to distinguish HPP from primary osteoporosis, as treatment and potential risks differ significantly. Inadvertent use of bisphosphonates can worsen hypomineralization and lead to atypical femoral fractures in HPP patients. Asfotase alfa enzyme replacement therapy, available since 2015, offers a targeted approach by replacing the enzyme defect, thereby mitigating symptoms. Heightened awareness among healthcare providers of HPP in adults holds the potential to enhance diagnosis rates and facilitate improved treatment outcomes for this highly treatable condition.[1] Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, Eighth Edition. ASBMR. Presentation: 6/2/2024

7740 Adult-Onset Hypophosphatasia Presenting As Low Alkaline Phosphatase Level Following Denosumab Use For Osteoporosis

Abstract Disclosure: J. Ocampo Mascaro: None. P. Wax: None. M. Al Mukaddam: Grant Recipient; Self; Ipsen, Incyte Research Funding. Background: Hypophosphatasia (HPP) is a genetic disease characterized by low activity of the tissue non-specific alkaline phosphatase. It has a broad range of clinical phenotypes, with adults usually presenting with an incidental low alkaline phosphatase (ALP) finding. Differentiating this from other causes of low ALP is challenging, especially when treating osteoporosis, as antiresorptive therapy such as denosumab and bisphosphonates may cause low ALP and carry the potential of increasing risk of atypical femoral fractures in patients with undiagnosed HPP. Clinical Case: A 64-year-old woman with history of nephrolithiasis, hypercalciuria, and osteoporosis (screening DXA lowest T-score -3.3 in femoral neck (FN)) but no prior fractures or dental issues was seen in clinic. Initial bloodwork showed normal range calcium, 25-OH vitamin D, and intact PTH. Pre-treatment ALP was 42 IU/L (N 39-117). Work up for secondary causes of osteoporosis was negative. Three yearly doses of IV zoledronic acid resulted in +4.2% BMD increase in lumbar spine (LS) but no changes in FN. She had no side effects but was found to have a decrease in ALP to 33 IU/L. This normalized back to 42 IU/L during a three-year drug holiday. Follow up DXA noted significant decline in BMD, hence denosumab was initiated. ALP decreased back to the 30-35 IU/L range. Fasting Vitamin B6 level (off B6 supplements for 1 week) was 23.4 ng/ml (N 2.1-21). Urine phosphoethanolamine was mildly elevated at 5 mmol/mol creatinine (N ≤4). Genetic testing evidenced a pathogenic ALPL variant (Exon 6, c.575T>C (p.Met192Thr), heterozygous) compatible with HPP. She had tolerated three doses of denosumab by then with BMD improvement of 5.0% in LS but no changes in FN and no atypical femoral fracture. Denosumab was stopped. Abaloparatide was started for osteoporosis with close monitoring for worsening hypercalciuria. Conclusion: This case demonstrates that adult HPP patients may present with subtle findings and lower range of normal ALP and may tolerate short courses of antiresorptive agents. There is a need for better screening and diagnostic criteria for adult HPP patients whose ALP levels may not be initially low before initiating antiresorptive drugs for osteoporosis. The risk of adverse effects from therapy will likely vary significantly based on the clinical severity/presentation of HPP as some adults with lower ALP ranges might be more susceptible to developing atypical femur fractures (1). Balancing atypical femur fracture risk with the benefits of antiresorptive treatment in decreasing risk of osteoporotic fractures highlights the need for better risk stratification to prevent undertreatment of osteoporosis. Reference: 1. Warren AM, Ebeling PR, Grill V, Seeman E, Sztal-Mazer S. Bilateral atypical femoral fractures during denosumab therapy in a patient with adult-onset hypophosphatasia. Endocrinol Diabetes Metab Case Rep. 2021 Sep 1;2021:21-0096. Presentation: 6/2/2024

7655 Genetic Mysteries Unveiled: Unusual Mutation Unraveled In Hypophosphatasia

Abstract Disclosure: A. Sharma: None. P. Katikaneni: None. S. Tanveer: None. K. Selk: None. Introduction: Hypophosphatasia (HPP), an inherited dental-osseous metabolic illness, is caused by inactivating mutations in the gene encoding the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP), which leads to a decrease in TNSALP enzymatic activity. Patients with HPP typically experience dental and skeletal mineralization issues as their most common symptoms; musculoskeletal abnormalities, numerous fractures, tiredness, and migraines are less common clinical presentations. Case Report: Our 75-year-old male patient initially presented 1.5 years prior following a spontaneous T12 vertebral fracture. A bone biopsy was performed and did not reveal evidence of malignancy. Testing for SPEP/UPEP was not consistent with myeloma, and thyroid testing was also unrevealing. His alkaline phosphatase level was consistently <40 IU/L since 1999. An evaluation was done for low alkaline phosphatase and secondary reasons for bone loss. This testing did not reveal vitamin D deficiency, parathyroid disease, hypophosphatemia, laboratory evidence of celiac disease, hypogonadism, or hypomagnesemia. A vitamin b6 level was markedly elevated at 119 ng/mL (ref range 2.1-21.7) while not on supplementation. He had noticed multiple fractures as a child, but he had written them off as minor injuries. He could clearly remember breaking his right wrist and clavicle. As an adult, he had rib fractures and a foot fracture. He was unable to recollect losing his primary teeth. Almost all his teeth are crowns or implants despite taking exceptional care of them. He did not have joint hypermobility. Genetic testing revealed the patient was heterozygous for an ALPL gene variant, c.876_882delinsT. It was a variant of undetermined significance at that time. His daughter also was found to have a low alkaline phosphatase and the same genetic test result. She also has experienced dental carries and joint pain. Our patient initiated teriparatide due to the need for more urgent therapy for extensive spinal surgery. He was later transitioned to asfotase alfa therapy and has not had any further fractures at the time of this report. Discussion: Hypophosphatasia is a rare inborn disorder due to mutations in the tissue nonspecific alkaline phosphatase gene ALPL and is characterized by low ALP levels. Affected individuals will also have elevated vitamin b6 levels and urinary phosphoethanolamine (PEA) levels. Early diagnosis of HPP is necessary to prevent bone fracture pain and improve quality of life. Inaccurate assessments can miss diagnoses of parents and other family members and even lead to lost opportunities to treat a patient with a substantial illness burden. This case highlights the mutation c.876_882delinsT as a likely pathologic variant in the ALPL gene. It also supports that patients with HPP can also respond to teriparatide treatment. Presentation: 6/2/2024

12472 Clinical Features Of Adult Hypophosphatasia And Correlation Between Laboratory Findings And Patient’s Symptomatology: A Retrospective Review At The Penn Bone Center

Abstract Disclosure: J. Ocampo Mascaro: None. R. Tran: None. S. Kallish: Consulting Fee; Self; Sanofi, Amicus. Research Investigator; Self; Ipsen, Sanofi, Incyte, Idorsia. Speaker; Self; Sanofi, Amicus. J. Berman: None. M. Al Mukaddam: Grant Recipient; Self; Ipsen, Incyte Research Funding. Hypophosphatasia (HPP) is an inborn error of metabolism resulting in low activity of the enzyme tissue non-specific alkaline phosphatase (ALP). It primarily involves bone and teeth mineralization. HPP has a wide clinical spectrum. It may present with rickets, fractures, failure to thrive, weakness, respiratory complications, and seizures in its more severe perinatal/infantile forms. Childhood forms tend to be milder but may have substantial musculoskeletal complications and early teeth loss. The clinical picture in adults is extremely variable too. It is usually mild with stress fractures and/or teeth loss in most individuals but can be debilitating in others due to musculoskeletal pain and recurrent fractures. This retrospective study describes the characteristics of 35 adult patients (ages 22-74 years) with HPP based on clinical presentation +/- molecular testing at the Penn Bone Center, located at a US-based large academic hospital. Out of 35 patients, 26 had an ALPL variant in genetic testing (12 pathogenic, 5 likely pathogenic, and 9 of uncertain significance), 3 were negative, and 6 did not undergo testing yet. ALP values ranged from 11 to 39 U/L (N: 38-126). Serum fasting vitamin B6 levels (off supplements) ranged from normal range to 18 times the upper limit of normal. Main symptoms reported were fractures (74%), arthralgias/myalgias (51%), diffuse bone pain (29%), atraumatic teeth loss (20%), muscle weakness (14%), and nephrolithiasis (9%). We found no significant negative correlation between ALP levels and number of symptoms (rs -0.026) or age of first symptom occurrence (rs 0.205). We also found no significant positive correlation between B6 elevation and number of symptoms (rs 0.236) or age of first symptom occurrence (rs -0.175). Six patients within our cohort had previous or current treatment with asfotase alfa. This is an FDA-approved therapy for the treatment of perinatal, infantile, and childhood HPP and has been used in adult patients if one of the presenting symptoms was before age 18 years. Response to therapy in our patient cohort was mixed. Three out of six patients discontinued treatment due to lack of clinical benefit. Continued research of the clinical characteristics of adult HPP is needed to better understand its natural history, markers of severity of disease, and how to determine who may potentially benefit from asfostase alfa therapy. Presentation: 6/1/2024

7077 A Rare Case Of Hypophosphatasia Presenting With Osteoporosis In A Young Female Patient- Diagnostic Delay In Hypophosphatasia

Abstract Disclosure: R. Abdelmasih: None. I. Ali: None. Introduction: Hypophosphatasia is a rare hereditary cause of secondary osteoporosis that affects 1/100.000 individuals. The rate of osteoporosis due to hypophosphatasia is probably underestimated as it likely goes undiagnosed in heterogeneous or milder cases. We present a case of Hypophosphatasia that presented initially with osteoporosis and went misdiagnosed for years with intermittent treatment of Bisphosphonate, luckily with no atypical fractures that prompted different course of management for osteoporosis. Case Presentation: A 44-year-old female with a history of rheumatoid arthritis and osteoporosis diagnosed 7 years prior and treated with intermittent bisphosphonate which was discontinued due to side effects including muscle aches and bone pains, presented to our endocrinology clinic to establish care for osteoporosis. Patient denied prior fractures. She reported jaw pain that is being evaluated by otolaryngology and a dentist. Family history is negative. DEXA scan showed Z scores of -1.1, -1.8, and -2.2 of the lumbar spine, femoral neck, and total femur respectively. Laboratory workup showed consistently low alkaline phosphatase (ALP) 25, 20, <20 U/L (ref range 34-123), elevated Vitamin B6 of 167 nmol/L (ref range 20-125), normal parathormone, calcium, phosphate, Vit D, magnesium, and liver enzymes. Genetic testing was sent for Tissue non-specific alkaline phosphatase (TNALP). Hypophosphatasia was diagnosed based on clinical and laboratory findings, patient was started on Asfotase alfa. Clinical course is to be followed. Discussion: Hypophosphatasia is a rare hereditary disorder due to mutation of (TNALP). In normal induvial, TNALP breaks down pyrophosphate to phosphate. It also plays a role in transferring Vit B6 into the tissues. Hence, clinical presentation due to impaired calcium-phosphate hemostasis is broad including musculoskeletal pain, pseudofractues, dental abscess, and neurological symptoms (due to accumulation of Vit B6). The clinical picture varies depending on the heterogenicity of ALPL mutation. Genetic testing is not mandated to establish diagnosis, clinical presentation and laboratory testing including low ALP and elevated Vit B6 are sufficient. However, genetic testing and counseling are recommended for the patient’s sake and their family members. Antiresorptive bone therapy is contraindicated due to a further decrease of bone turnover with reported cases of increased risk of atypical femur fracture, Bone density is reported to improve with enzyme replacement therapy with bone-targeting recombinant alkaline phosphatase (Asfotase Alpha), if further treatment is needed, anabolic treatment is preferred. We present this case to shed light on such a rare disease to decrease rates of underdiagnosis or delayed diagnosis in young patients presenting with osteoporosis for prompt treatment and improved quality of life of these patients. Presentation: 6/2/2024

Tissue nonspecific alkaline phosphatase deficiency impairs Purkinje cell development and survival in a mouse model of infantile hypophosphatasia

Loss-of-function mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene can result in hypophosphatasia (HPP), an inherited multi-systemic metabolic disorder that is well-known for skeletal and dental hypomineralization. However, emerging evidence shows that both adult and pediatric patients with HPP suffer from cognitive deficits, higher measures of depression and anxiety, and impaired sensorimotor skills. The cerebellum plays an important role in sensorimotor coordination, cognition, and emotion. To date, the impact of TNAP mutation on the cerebellar circuitry development and function remains poorly understood. The main objective of this study was to investigate the roles of TNAP in cerebellar development and function, with a particular focus on Purkinje cells, in a mouse model of infantile HPP. Male and female wild type (WT) and TNAP knockout (KO) mice underwent behavioral testing on postnatal day 13–14 and were euthanized after completion of behavioral tests. Cerebellar tissues were harvested for gene expression and immunohistochemistry analyses. We found that TNAP mutation resulted in significantly reduced body weight, shorter body length, and impaired sensorimotor functions in both male and female KO mice. These developmental and behavioral deficits were accompanied by abnormal Purkinje cell morphology and dysregulation of genes that regulates synaptic transmission, cellular growth, proliferation, and death. In conclusion, inactivation of TNAP via gene deletion causes developmental delays, sensorimotor impairment, and Purkinje cell maldevelopment. These results shed light on a new perspective of cerebellar dysfunction in HPP.

Reversal of heavy arterial calcification in a rat model of chronic kidney disease using targeted ethylene diamine tetraacetic acid-loaded albumin nanoparticles.

Elastin degradation and severe calcification in the medial layer of the vessel wall, known as medial arterial calcification (MAC), is typical in the aging population and patients with metabolic disorders, such as diabetes and chronic kidney disease (CKD). We have previously reported that ethylene diamine tetraacetic acid (EDTA) delivery to the site of calcification can be achieved by tagging nanoparticles with an elastin antibody that recognizes explicitly damaged elastin, and such systemic therapy can remove focal calcium deposits from the calcified arteries in CKD rodent model. The current study aims to test whether heavy calcification seen throughout arterial tree and kidneys in CKD can be reversed with nanoparticle therapy. Thirty healthy male Sprague-Dawley rats weighing approximately 300 g, were placed on an adenine diet for 21 non-consecutive days to induce kidney failure, followed by daily vitamin D3 (VitD3) injections for 4 sequential days to cause severe calcification throughout the cardiovascular system and kidneys. DiR-dye loaded and elastin antibody conjugated albumin nanoparticles were used to confirm the targeting of nanoparticles to the calcification area. The rats were divided into two groups for targeted removal of calcification starting at day 7 of the last doses of VitD3. The experimental group received biweekly IV injections of anti-elastin antibody conjugated EDTA loaded human serum albumin nanoparticles (EDTA-HSA-El-Ab NPs), while the sham controls received blank nanoparticles (Blank-HSA-El-Ab NPs) (5 injections in total). Micro-computed tomography (microCT) was used to analyze the extent of calcification. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry studies were performed for osteogenic markers, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), and tissue non-specific alkaline phosphatase (TNAP). For comparison, aortic ring organ cultures from healthy rats were treated with high phosphate to induce calcification in vitro, and then they were treated with EDTA. Human calcified femoral arteries were also treated ex vivo with EDTA-HSA-EL-Ab NPs to test if nanoparticles remove heavy calcification. EDTA-loaded nanoparticles that specifically target degraded elastin reversed existing heavy mineral deposits in arteries, as per elemental calcium analysis (124.161±34.410 µg Ca per mg of the dry aorta in Blank-HSA-El-Ab NPs vs. 100.520±19.131 µg in EDTA-HSA-El-Ab NPs group, P=0.04) and microCT (object volume, 129.001±37.785 vs. 29.815±24.169 mm3, P=0.0005). The reversal of aortic calcification was accompanied by a significant reduction of bone-associated mRNA expression of BMP2 and RUNX2 (P=0.01). Immunohistochemistry studies corroborated RT-PCR results, showing a reduction of BMP2 and RUNX2 stains in the vessel wall. The rat aortic ring culture study also showed similar results, where osteogenic genes (BMP2, RUNX2) and proteins (BMP2, RUNX2, TNAP) were suppressed upon reversal of calcification with EDTA (P=0.001). We also show ex vivo reversal of human femoral artery calcification by microCT (calcium intensity: untreated, 57.721±28.551 vs. day 6 of treatment, 5.441±3.615, P=0.01) by EDTA nanoparticle therapy. This is the first study showing the removal of calcium from heavily calcified arteries by using intravenous targeted EDTA therapy. Such therapy also reversed vascular smooth muscle cell osteoblastic transition and apoptosis in the arterial tissue, thereby potentially creating an environment for suitable tissue repair.

Enzyme replacement therapy for hypophosphatasia-The current paradigm.

Hypophosphatasia (HPP) is a rare, inherited, and systemic disorder characterized by impaired skeletal mineralization and low tissue nonspecific serum alkaline phosphatase (TNSALP) activity. It is caused by either autosomal recessive or dominant-negative mutations in the gene that encodes TNSALP. The phenotype of HPP is very broad including abnormal bone mineralization, disturbances of calcium and phosphate metabolism, pain, recurrent fracture, short stature, respiratory impairment, developmental delay, tooth loss, seizures, and premature death. Other than supportive care, there has been no disease-specific treatment available for those with HPP. Asfotase alfa is a fully humanized, recombinant enzyme replacement therapy for the management of HPP. It is available in several countries for the treatment of the more severe forms of HPP, namely perinatal and infantile HPP. This review will summarize the preclinical data on asfotase alfa and highlight the data from clinical trials and case reports. These data show the transformative nature of asfotase alfa when administered as part of an interdisciplinary treatment model.

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.

Would three BMPs at low concentration be better than one at high concentration? An experimental study with rat osteoprogenitor cells.

Bone morphogenetic proteins (BMPs) are used in clinical practice for stimulation of bone formation, but often evoke serious complications. Recent studies demonstrated that BMPs involved in early stages of bone formation are species specific. In cattle dominate BMP7, growth differentiation factor 5 (GDF5) and NEL-like protein 1 (NELL1) while in rats BMP2, BMP5 and BMP6. The purpose of the study was to compare the action of the species specific BMPs on the osteoprogenitor cells. Thus, rat osteoprogenitor cells were exposed to one BMP in a high dose and three of them at 1/3 of the former. Isolated rat osteoprogenitor cells were treated in culture with different concentrations of BMP2, BMP5 and BMP6 or with lower concentration of combinations of these cytokines. Activity of alkaline phosphatase, calcium deposition and mRNA level for transcription factor SP7 (osterix) and tissue non-specific alkaline phosphatase (TNAP) served as indicators of BMPs effect. BMPs stimulated all studied parameters in comparison with control cultures, but no statistically significant differences were observed between the action of a large dose of one cytokine and a combination of cytokines given at lower concentrations. Three BMPs used in a low dose exert similar effect as the one used at high dose. Since the BMPs stimulate different receptors and activate different signaling pathways the use of the mixture of properly chosen BMPs at low concentration may give better results than the single one at high concentration and may avoid untoward effects.

Alpha-synuclein aggregates are phosphatase resistant

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp–Lantos bodies in Parkinson’s disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

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.

Rare Genetic Developmental Disabilities: Mabry Syndrome (MIM 239300) Index Cases and Glycophosphatidylinositol (GPI) Disorders.

The case report by Mabry et al. (1970) of a family with four children with elevated tissue non-specific alkaline phosphatase, seizures and profound developmental disability, became the basis for phenotyping children with the features that became known as Mabry syndrome. Aside from improvements in the services available to patients and families, however, the diagnosis and treatment of this, and many other developmental disabilities, did not change significantly until the advent of massively parallel sequencing. As more patients with features of the Mabry syndrome were identified, exome and genome sequencing were used to identify the glycophosphatidylinositol (GPI) biosynthesis disorders (GPIBDs) as a group of congenital disorders of glycosylation (CDG). Biallelic variants of the phosphatidylinositol glycan (PIG) biosynthesis, type V (PIGV) gene identified in Mabry syndrome became evidence of the first in a phenotypic series that is numbered HPMRS1-6 in the order of discovery. HPMRS1 [MIM: 239300] is the phenotype resulting from inheritance of biallelic PIGV variants. Similarly, HPMRS2 (MIM 614749), HPMRS5 (MIM 616025) and HPMRS6 (MIM 616809) result from disruption of the PIGO, PIGW and PIGY genes expressed in the endoplasmic reticulum. By contrast, HPMRS3 (MIM 614207) and HPMRS4 (MIM 615716) result from disruption of post attachment to proteins PGAP2 (HPMRS3) and PGAP3 (HPMRS4). The GPI biosynthesis disorders (GPIBDs) are currently numbered GPIBD1-21. Working with Dr. Mabry, in 2020, we were able to use improved laboratory diagnostics to complete the molecular diagnosis of patients he had originally described in 1970. We identified biallelic variants of the PGAP2 gene in the first reported HPMRS patients. We discuss the longevity of the Mabry syndrome index patients in the context of the utility of pyridoxine treatment of seizures and evidence for putative glycolipid storage in patients with HPMRS3. From the perspective of the laboratory innovations made that enabled the identification of the HPMRS phenotype in Dr. Mabry's patients, the need for treatment innovations that will benefit patients and families affected by developmental disabilities is clear.

Identifying alkaline phosphatase inhibitory potential of cyclooxygenase-2 inhibitors: Insights from molecular docking, MD simulations, molecular expression analysis in MCF-7 breast cancer cell line and in vitro investigations

Alkaline phosphatases (APs, EC 3.1.3.1) belong to a superfamily of biological macromolecules that dephosphorylate many phosphometabolites and phosphoproteins and their overexpression is intricated in the spread of cancer to liver and bones, neuronal disorders including Alzheimer's disease (AD), inflammation and others. It was hypothesized that cyclooxygenase-2 (COX-2) selective inhibitors may possess anti-APs potential and may be involved in anticancer proceedings. Three COX-2 inhibitors including nimesulide, piroxicam and lornoxicam were evaluated for the inhibition of APs using in silico and in vitro methods. Molecular docking studies against tissue nonspecific alkaline phosphatase (TNAP) offered the best binding affinities for nimesulide (−11.14 kcal/mol) supported with conventional hydrogen bonding and hydrophobic interactions. MD simulations against TNAP for 200 ns and principal component analysis (PCA) reiterated the stability of ligand-receptor complexes. Molecular expression analysis of TNAP enzyme in the breast cancer cell line MCF-7 exhibited 0.24-fold downregulation with 5 μM nimesulide as compared with 0.26-fold standard 10 μM levamisole. In vitro assays against human placental AP (hPAP) displayed potent inhibitions of these drugs with IC50 values of 0.52 ± 0.02 μM to 3.46 ± 0.13 μM and similar results were obtained for bovine intestinal AP (bIAP). The data when generalized collectively emphasizes that the inhibition of APs by COX-2 inhibitors provides another target to work on the development of anticancer drugs.

P074 Diagnostic Journeys of Patients with Hypophosphatasia

Abstract Background/Aims Hypophosphatasia (HPP) is an ultra-rare genetic disorder characterised by low activity of tissue non-specific alkaline phosphatase (TNAP). Due to heterogeneity of clinical presentation and lack of definitive biochemical testing, misdiagnosis and diagnostic delay are common. Methods We used information reported by HPP patients to the Rare and Undiagnosed Diseases Study (RUDY) online registry (www.rudystudy.org). We included patients with a self-reported diagnosis of HPP and information on at least one of our outcomes. We described their diagnostic journey, including diagnostic delays, initial symptoms, incorrect diagnoses and healthcare professionals seen. Results 16 participants with a self-reported diagnosis of HPP were included in the analysis with 14/16 female. Of those reporting the date of initial symptom onset, 9/14 participants had symptom onset before the age of 18, whereas five had symptom onset in adulthood. The median time from symptom onset to HPP final diagnosis was 26.5 years (N = 8, IQR 11.2 to 34.6). From the first GP visit related HPP to final diagnosis the median time was 9.5 years (N = 6, IQR 6.2 to 11.1), and from the first hospital visit related to HPP, the median time to final diagnosis was 3.9 years (N = 10, IQR 0.8 to 10.5). Among participants reporting initial symptoms (N = 13), 16 categories of initial symptom were reported, with a median of two initial symptom categories per participant (IQR 1 to 2). The most prevalent initial symptom was pain (10/13, 77%). Of these 10 participants, four had lower limb pain, one had upper limb pain, one had back pain, and five had pain of unspecified location. Skeletal deformity at birth, dental manifestations, joint swelling, and infantile feeding problems were each present in 2/13 participants (15%). Among participants reporting whether they had received other diagnoses prior to that of HPP (N = 13), nine participants (75%) received at least one other diagnosis, with diagnoses of 10 different conditions reported across the cohort. The most common other diagnosis was fibromyalgia (4/13, 31%), followed by hypermobility (2/13, 15%). 21 different types of healthcare professionals were seen prior to final diagnosis. General Practitioners were the most commonly consulted (9/14, 64%). This was followed by allied health professionals (8/14, 57%). Of the eight participants seeing allied health professionals, all eight saw physiotherapists, and 4 (29% of total) also saw at least one other allied health professional. Other types of healthcare professional seen by multiple responders included orthopaedic surgeons (5/14, 36%), rheumatologists (5/14, 36%), geneticists (4/14, 29%), endocrinologists (3/14, 21%), metabolic specialists (3/14, 21%) and dentists (2/14, 14%). Conclusion Patients typically present with a cluster of symptoms, often including pain, and are often seen by general practitioners and physiotherapists before diagnosis. Increasing awareness of HPP in these professions may improve early recognition of the disease. Disclosure D. Marsden: None. R. Ridley: None.

Detection of hypophosphatasia in hospitalised adults in rheumatology and internal medicine departments: a multicentre study over 10 years

IntroductionHypophosphatasia (HPP) is a rare genetic disease caused by loss-of-function mutations in the ALPL gene encoding the tissue non-specific alkaline phosphatase (ALP). Mild HPP is usually misdiagnosed in adult age....

Mechanical stretch leads to increased caveolin-1 content and mineralization potential in extracellular vesicles from vascular smooth muscle cells

BackgroundHypertension-induced mechanical stress on vascular smooth muscle cells (VSMCs) is a known risk factor for vascular remodeling, including vascular calcification. Caveolin-1 (Cav-1), an integral structural component of plasma membrane invaginations, is a mechanosensitive protein that is required for the formation of calcifying extracellular vesicles (EVs). However, the role of mechanics in Cav-1-induced EV formation from VSMCs has not been reported.ResultsExposure of VSMCs to 10% mechanical stretch (0.5 Hz) for 72 h resulted in Cav-1 translocation into non-caveolar regions of the plasma membrane and subsequent redistribution of Cav-1 from the VSMCs into EVs. Inhibition of Rho-A kinase (ROCK) in mechanically-stimulated VSMCs exacerbated the liberation of Cav-1 positive EVs from the cells, suggesting a potential involvement of actin stress fibers in this process. The mineralization potential of EVs was measured by incubating the EVs in a high phosphate solution and measuring light scattered by the minerals at 340 nm. EVs released from stretched VSMCs showed higher mineralization potential than the EVs released from non-stretched VSMCs. Culturing VSMCs in pro-calcific media and exposure to mechanical stretch increased tissue non-specific alkaline phosphatase (ALP), an important enzyme in vascular calcification, activity in EVs released from the cells, with cyclic stretch further elevating EV ALP activity compared to non-stretched cells.ConclusionOur data demonstrate that mechanical stretch alters Cav-1 trafficking and EV release, and the released EVs have elevated mineralization potential.

CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner

BackgroundClinical and experimental studies have shown that the myocardial inflammatory response during pathological events varies between males and females. However, the cellular and molecular mechanisms of these sex differences remain elusive. CD73/adenosine axis has been linked to anti-inflammatory responses, but its sex-specific cardioprotective role is unclear. The present study aimed to investigate whether the CD73/adenosine axis elicits sex-dependent cardioprotection during metabolic changes and myocarditis induced by hypobaric hypoxia.MethodsFor 7 days, male and female mice received daily injections of the CD73 inhibitor adenosine 5′- (α, β-methylene) diphosphate (APCP) 10 mg/kg/day while they were kept under normobaric normoxic and hypobaric hypoxic conditions. We evaluated the effects of hypobaric hypoxia on the CD73/adenosine axis, myocardial hypertrophy, and cardiac electrical activity and function. In addition, metabolic homeostasis and immunoregulation were investigated to clarify the sex-dependent cardioprotection of the CD73/adenosine axis.ResultsHypobaric hypoxia-induced cardiac dysfunction and adverse remodeling were more pronounced in male mice. Also, male mice had hyperactivity of the CD73/adenosine axis, which aggravated myocarditis and metabolic shift compared to female mice. In addition, CD73 inhibition triggered prostatic acid phosphatase ectonucleotidase enzymatic activity to sustain adenosine overproduction in male mice but not in female mice. Moreover, dual inhibition prostatic acid phosphatase and CD73 enzymatic activities in male mice moderated adenosine content, alleviating glycolytic shift and proinflammatory response.ConclusionThe CD73/adenosine axis confers a sex-dependent cardioprotection. In addition, extracellular adenosine production in the hearts of male mice is influenced by prostatic acid phosphatase and tissue nonspecific alkaline phosphatase.