Biology subjects

Millan, J. L.

Publications and source records attributed to Millan, J. L..

5 recordsLinked to original sources

bioRxiv2026

TNAP and PHOSPHO1 function synergistically to afford critical control over the mineralisation of the postnatal murine skeleton

Biomineralisation is essential for skeletal integrity, yet the synergistic roles of tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 in postnatal bone mineralisation remain poorly defined. To decipher this, we generated a novel murine model in which Alpl was deleted in Prx1- expressing cells (AlplPrx1/Prx1) in mice with a global Phospho1-/- deficiency to overcome the perinatal lethality that arises upon dual global deletion. Using a multi-modal approach to spatially phenotype the limbs of these animals, we reveal mice lacking both TNAP and PHOSPHO1 exhibit a distinct lack of mineralisation and altered anatomical structure at postnatal day 1 (PN1) and 3-weeks of age. Although viable, these mice did not thrive due to their reduced size, thus further investigations were conducted on mice with a heterozygous deletion of TNAP (Alplwt/Prx1;Phospho1-/-). Although smaller than wild-types at PN1 and 3 weeks old, these mice did not display the gross limb deformations observed in the homozygous animals and the single, functioning Alpl allele rescued the loss of biomineralisation observed following dual phosphatase deletion. At 6-weeks of age, compromised epiphyses and metaphyses were only seen in AlplPrx1/Prx1 animals. Further, we found that tibial geometry and porosity was significantly altered by Phospho1 deletion (Phospho1-/-), which was compounded in the Alplwt/Prx1;Phospho1-/- mice and linked to alterations in collagen configuration, matrix mineralisation and growth plate deformities. Together, our findings establish the mechanistic framework for TNAP and PHOSPHO1 in permissive biomineralisation, providing critical insights into this fundamental process. Significance StatementBiomineralisation is essential for skeletal development and is critically dependent on phosphatases that release inorganic phosphate for hydroxyapatite formation. Our study investigates the dual role of PHOSPHO1 and TNAP in this process, using a novel murine knockout model. Deletion of both enzymes results in complete loss of bone mineralisation, demonstrating their critical synergistic function. Further, we show that PHOSPHO1 and TNAP exhibit distinct, spatially-restricted functions in the tibia and thus enhances our understanding of the fundamentals processes underpinning biomineralisation. These findings also have clinical relevance as they have the potential to inform on treatment strategies for hypo- and hyper-mineralised pathologies.

developmental biology↗

bioRxiv2024

Subendothelial Calcification Increases Surface Roughness and Promotes Lipid Deposition into the Arterial Wall

ObjectiveWe have previously demonstrated that subendothelial calcification accelerates atherosclerosis in mice. This study addresses a mechanism by which subendothelial calcifications can increase low-density lipoprotein (LDL) uptake into the arterial wall. MethodsMice overexpressing tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells (eTNAP mice) were used as a model of calcification. Calcification and atherosclerosis were detected by micro-computed tomography (micro-CT) and histology. The endothelial roughness was characterized by surface metrology. A fluid-structure interaction model was used to calculate wall shear stress (WSS). The uptake of fluorescent LDL was traced in vitro and in vivo. Human arteries were assessed for the prevalence of internal elastic lamina (IEL) calcification. ResultseTNAP mice developed more severe aortic atherosclerosis than controls on the LDL receptor mutant background (p<0.01). Subendothelial calcifications in eTNAP mice were confirmed by micro-CT. An increase in aortic surface area roughness, including the height, volume, and steepness parameters, was observed in eTNAP mice compared to controls (p<0.01). Calcifications affected near-wall hemodynamics, creating pockets of reduced WSS. Endothelial cells cultured on rough surfaces showed increased LDL uptake compared to cells cultured on smooth collagen (p<0.0001). Fluorescent LDLs were traced to subendothelial calcifications in eTNAP mice but not in controls. In humans, IEL calcification was prevalent in older adults and inversely correlated with arterial diameter (p<0.05). ConclusionSubendothelial calcification is sufficient to perturb near-wall hemodynamics, creating localized areas of reduced WSS, consistent with increased LDL uptake near calcified lesions. Subendothelial calcification may represent an alternative or concurrent mechanism for the initiation of atherosclerosis. Research perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIWe tested a novel hypothesis that subendothelial microcalcification can initiate atherosclerosis. C_LIO_LIThe study demonstrated that micron-sized subendothelial calcifications, induced by the overexpression of tissue-nonspecific alkaline phosphatase in the endothelium, are sufficient to perturb local hemodynamics, creating pockets of low wall shear stress, consistent with an increase in low-density lipoprotein uptake and deposition into the arterial wall in juxtaposition to calcified lesions. C_LI What are the clinical implications?O_LIWe suggest that calcification of internal elastic lamina of medium-sized arteries may represent early lesions initiating atherosclerosis; however, the epidemiologic evidence for this theory is currently unavailable. C_LI

physiology↗

bioRxiv2021

Differential Expression of Alkaline Phosphatase and PHOSPHO1 in Bone from a Murine Model of Chronic Kidney Disease

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities; a condition known as renal osteodystrophy (ROD). While Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are recognized to be critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both wild-type and Phospho1 knockout (P1KO) mice using dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (p < 0.05) but decreased in trabecular bone (p < 0.05). These changes were accompanied by decreased TNAP (p < 0.01) and increased PHOSPHO1 (p < 0.001) expression in wild-type CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expression to be down-regulated in response to phosphate and PTH. While matrix mineralization was increased with phosphate (Pi), it decreased with PTH and FGF23 had no effect. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism in an attempt to protect the bone from hypomineralization. We propose that increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

cell biology↗

bioRxiv2021

Brain endothelial cell tissue-nonspecific alkaline phosphatase (TNAP) activity promotes maintenance of barrier integrity via the ROCK pathway

Blood-brain barrier (BBB) dysfunction is a key feature in many neuroinflammatory diseases. Yet, no therapies exist to effectively mitigate BBB dysfunction. A strategy to bridge this knowledge gap requires an examination of proteins localized to brain microvascular endothelial cells (BMECs) and evaluating their role in preserving barrier integrity. Tissue-nonspecific alkaline phosphatase (TNAP) is highly abundant in brain microvascular endothelial cells (BMECs); however, its function in BMECs remains unclear. We hypothesized that a loss or inhibition of TNAP activity on BMECs would impair barrier integrity through increased cytoskeletal remodeling driven by the Rho-associated protein kinase (ROCK) pathway. First, we examined barrier integrity in hCMEC/D3 cells treated with a TNAP inhibitor (TNAPi) and in primary BMECs (pBMECs) via the conditional deletion of TNAP in endothelial cells. Our results showed that both pharmacological inhibition and genetic conditional loss of TNAP significantly worsened endothelial barrier integrity compared to controls. Next, we examined the mechanisms through which TNAP activity exerts a protective phenotype on BMECs. Our results showed that hCMEC/D3 cells treated with TNAPi displayed remarkable phalloidin and vimentin cytoskeletal remodeling compared to control. We then examined the role of ROCK, a key player in cytoskeletal remodeling. Our results showed that TNAPi increased the expression of ROCK 1/2. Furthermore, inhibition of ROCK 1/2 with fasudil mitigated TNAPi-induced and VE-cKO barrier dysfunction. Collectively, our results support a novel mechanism through which loss of TNAP activity results in cerebrovascular dysfunction, and selective modulation of TNAP activity in BMECs may be a therapeutic strategy to improve BBB function.

neuroscience↗

bioRxiv2020

PHOSPHO1, a novel skeletal regulator of insulin resistance and obesity

The skeleton is recognised as a key endocrine regulator of metabolism. Here we show that mice lacking the bone mineralization enzyme PHOSPHO1 (Phospho1-/-) exhibited improved basal glucose homeostasis and resisted high-fat-diet induced weight gain and diabetes. The metabolic protection in Phospho1-/- mice was manifested in the absence of altered levels of osteocalcin. Osteoblasts isolated from Phospho1-/- mice were enriched for genes associated with energy metabolism and diabetes; Phospho1 both directly and indirectly interacted with genes associated with glucose transport and insulin receptor signalling. Canonical thermogenesis via brown adipose tissue did not underlie the metabolic protection observed in adult Phospho1-/- mice. However, the decreased serum choline levels in Phospho1-/- mice were normalized by feeding a 2% choline rich diet resulting in a normalization in insulin sensitivity and fat mass. This study identifies PHOSPHO1 as a potential therapeutic target for the treatment of obesity and diabetes.

physiology↗