Biology subjects

Rajpar, I.

Publications and source records attributed to Rajpar, I..

7 recordsLinked to original sources

bioRxiv2026

Phosphocitrate Is Superior to Pyrophosphate in Preventing Soft Connective Tissue Calcification in a Mouse Model of Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is a rare inherited disorder characterized by progressive ectopic calcification of soft connective tissues, including skin, arteries, and eyes, leading to significant morbidity. PXE results from loss of functional ABCC6, a liver specific ATP efflux conduit. Reduced ATP release into the circulation limits its conversion into AMP and the mineralization inhibitor pyrophosphate (PPi). Consequently, low plasma PPi levels drive ectopic calcification in PXE. Although oral PPi supplementation can inhibit ectopic calcification in Abcc6-/- mice, impractically high doses are needed, due to its rapid hydrolysis in the gastrointestinal tract. Here, we evaluated phosphocitrate, an exceedingly more potent mineralization inhibitor, in vitro and in Abcc6-/- mice. In ATDC5 cells, 1 {micro}M phosphocitrate significantly inhibited mineralization following induction, comparable to approximately tenfold higher concentrations of PPi. In vivo, daily intraperitoneal administration of phosphocitrate (4.7 {micro}mol/kg bw) markedly reduced calcification in muzzle skin and kidneys, whereas an at least fivefold higher dose of PPi was needed to achieve a similar effect. Oral administration required substantially higher doses of both compounds ([~]2.4 mmol/kg bw), but PC remained more effective than PPi at inhibiting soft tissue calcification in Abcc6-/- mice. Importantly, unlike PPi, oral phosphocitrate did not adversely affect skeletal strength or stiffness, even at supraphysiological doses. In summary, phosphocitrate is a more potent inhibitor of ectopic calcification than PPi in Abcc6-/- mice. While optimization of oral delivery remains necessary, its increased potency supports the potential of alternative administration routes, including subcutaneous delivery, as a practical therapeutic strategy for PXE.

pharmacology and toxicology↗

bioRxiv2025

ABCC6 and ANK regulate extracellular homeostasis of pyrophosphate and citrate and affect mineral deposition in bones and soft connective tissues

ABCC6 and ANK are integral membrane proteins involved in the efflux of specific organic anions. ABCC6, primarily expressed in the liver, regulates circulating levels of the mineralization inhibitor pyrophosphate (PPi). In contrast, ANK is widely expressed and plays a dual role, maintaining extracellular PPi homeostasis and, notably, mediating cellular citrate efflux. We studied how both proteins affected extracellular metabolite levels, ectopic calcification, and bone homeostasis. We observed that plasma PPi was reduced by [~]36% in Ankank/ankmice and [~]60% in Abcc6-/- mice. However, plasma citrate levels depended primarily on ANK, dropping [~]75% in Ankank/ank and double mutants, but remaining unchanged in Abcc6-/- mice. MicroCT revealed extreme ectopic calcification in double mutants, far exceeding either single knockout, affecting muzzle skin and ear cartilage. Oral citrate was bioavailable and, at high doses, prevented soft tissue calcification in Abcc6-/- mice, suggesting a systemic protective role. In bone, ANK was essential for incorporating both PPi and citrate, while ABCC6 mainly affected PPi. ANK deficiency led to reduced trabecular volume, cortical thickness, cortical area fraction, and mineral density, with more pronounced effects in males. Biomechanical testing showed decreased ultimate moment, bending rigidity, and energy in ANK-deficient femora, alongside increased post-yield displacement, indicating compensatory matrix changes. Collectively, our findings identify ANK as a dual regulator of PPi and citrate, with a previously unrecognized role in preventing soft tissue calcification. This study positions ANK as a potential therapeutic target for mineralization disorders like pseudoxanthoma elasticum (caused by ABCC6 deficiency) and conditions of low bone mineral density like osteoporosis.

developmental biology↗

bioRxiv2025

High-dose oral pyrophosphate inhibits connective tissue calcification in Abcc6 null mice but affects bone structure

Pseudoxanthoma elasticum is a rare inherited disorder marked by abnormal calcium phosphate deposition in soft connective tissues, particularly the skin, arteries, and eyes. It is caused by inactivating mutations in the ABCC6 gene, which encodes a hepatic efflux transporter. Loss of ABCC6 function leads to reduced plasma levels of pyrophosphate, a key inhibitor of calcification, thereby promoting ectopic mineralization. Oral pyrophosphate therapy has emerged as a potential treatment, but its effectiveness is uncertain. Most ingested pyrophosphate is hydrolyzed in the gut to inorganic phosphate, which may worsen calcification. Moreover, its impact on mineralized tissues remains largely unexplored. Abcc6-/- mice closely mimic human pseudoxanthoma elasticum and are widely used in preclinical studies. Although patients are most concerned about ocular complications, eye calcification is rarely assessed in translational studies using Abcc6-/- mice. Using microcomputed tomography we found that ectopic calcification at the ciliary margin is a reliable marker of ocular disease progression in these mice. Administering pyrophosphate in drinking water at concentrations up to 90 mM did not increase calcification in skin or eyes. However, only very high doses effectively prevented ectopic calcification - doses that would equate to an impractical 2.5 g/kg/day of disodium pyrophosphate in humans. These high doses also led to pyrophosphate accumulation in bone and negatively affected bone structure and strength. In summary, only supraphysiological doses of orally administered pyrophosphate inhibited ectopic calcification in Abcc6-/- mice, but these doses are not feasible for human use and may compromise bone function. These data are especially important considering the currently ongoing clinical trial evaluating the safety and efficacy of oral pyrophosphate administration as a treatment for pseudoxanthoma elasticum. LAY SUMMARYPseudoxanthoma elasticum (PXE) is a rare inherited mineralization disorder caused by the absence of functional ABCC6, a liver-expressed protein. This deficiency leads to reduced plasma levels of pyrophosphate, a key inhibitor of mineralization, resulting in abnormal calcium phosphate deposition in the skin, eyes, and blood vessels. Oral pyrophosphate has been proposed as a therapeutic strategy for PXE, and a clinical trial evaluating its efficacy recently began in France. In a PXE mouse model, we show that only very high oral doses of pyrophosphate are effective, but these doses impair bone quality and are not suitable for human use.

pathology↗

bioRxiv2025

Osteoblast-derived Nerve Growth Factor is Required for Skeletal Adaptation to Mechanical Load and the Osteoanabolic Effect of Gambogic Amide in Mice

In adult mice, new bone accrual following mechanical load is mediated by the neurotrophin nerve growth factor (NGF) that is expressed by osteoblasts on the bone surface. NGF can bind to its high affinity receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), on peripheral sensory nerves resident in bone and support new bone formation. However, the osteoanabolic therapeutic potential of NGF-TrkA signaling to repair bone is limited due to the long-lasting thermal and mechanical hyperalgesia induced by administration of NGF in mice and humans. Here, we investigated whether 1) mature osteoblasts are the primary source of NGF required for bone accrual following loading, and 2) a small molecule TrkA receptor agonist - gambogic amide - can harness the downstream osteoanabolic potential of NGF-TrkA signaling in the absence of endogenous NGF. Loss of Ngf transcription in mature osteoblasts did not appear to affect bone structure or bone mass in adulthood. However, Ngf knockout mice significantly reduced periosteal bone accrual and osteogenic Wnt transcription in response to loading compared to wildtype mice. Intraperitoneal injection of gambogic amide prior to loading was unable to produce its osteoanabolic effects in Ngf knockout mice, suggesting that gambogic amide primarily functions in collaboration with endogenous NGF in bone. In total, our study reveals an important role for osteoblastic NGF in the skeletal adaptation of bone to mechanical forces.

bioengineering↗

bioRxiv2025

The Effect of Celecoxib and muMab911 on Strain Adaptive Bone Remodeling and Fracture Repair in Female Mice: Implications for Rapidly Progressive Osteoarthritis

Debilitating pain is the primary clinical feature of osteoarthritis (OA) that drives the enormous healthcare costs. Osteoarthritis-related pain is often treated with non-steroidal anti-inflammatory drugs (NSAIDs), which effectively relieve pain and inflammation by inhibition of prostaglandin synthesis. Antibodies directed against nerve growth factor (NGF) were tested some time ago as an alternative potential analgesic for musculoskeletal pain, including osteoarthritis-related pain. Unfortunately, clinical development of these drugs was put on hold due to adverse outcomes - primarily rapidly progressive osteoarthritis. Both prostaglandin synthesis and NGF have been implicated as critical mediators of strain adaptive bone remodeling, which may play a role in rapid osteoarthritis progression. Therefore, this study was designed to investigate the effects of celecoxib, an NSAID, and muMab911, an anti-NGF antibody, as well as the combination therapy on strain adaptive bone remodeling, bone mass and geometry, and bone healing in a murine model. Adult female C57BL/6J mice received celecoxib through drinking water, up to 3 IP injections of muMab911, or both treatments over a period of two weeks. As expected, all treatments were effective for relieving injury-associated pain. Consistent with previous studies, we found that celecoxib alone and in combination with muMab911 impaired periosteal load-induced bone formation induced by axial forelimb compression. Furthermore, both treatments had minimal effects on osteoblast and osteocyte populations, bone structural and material properties, and cortical and trabecular bone mass. Mice subjected to damaging axial forelimb compression did not have impaired healing or callus morphology due to treatment. Nonetheless, both drugs decreased NGF expression during fracture healing. In total, our results indicate that these medications both suppress NGF expression and load-induced bone formation which may support rapid OA disease progression by diminishing strain adaptive bone remodeling in subchondral bone of OA patients.

bioengineering↗

bioRxiv2022

Toll-like receptor 4 signaling in osteoblasts is required for load-induced bone formation in mice

During skeletal development, expression of nerve growth factor (NGF) leads to the survival of afferent sensory nerves that express neurotrophic tyrosine kinase receptor type 1 (TrkA), the high affinity receptor for NGF. In adulthood, NGF is expressed by mature osteoblasts following mechanical loading and signals through TrkA receptors in resident sensory nerves to support load-induced bone formation. However, the regulation of NGF in osteoblasts following loading is not well understood. In this study, we sought to determine whether osteoblastic expression of toll-like receptor 4 (TLR4), a key receptor in the NF-{kappa}B signaling pathway, is required to initiate NGF-TrkA signaling to support skeletal adaptation following mechanical loading. First, we observed that NF-{kappa}B inhibition reduces NGF expression induced by axial forelimb compression. Moreover, we observed that TLR4+ periosteal cells are increased after mechanical loading. Therefore, we generated a novel mouse model in which Tlr4 is selectively removed from the mature osteoblast lineage. Although Tlr4 conditional knockout mice have normal skeletal mass and strength in adulthood, the loss of TLR4 signaling results in significant reductions in periosteal lamellar bone formation following axial forelimb compression. Furthermore, we demonstrated that the upregulation of Ngf following application of fluid shear stress to calvarial osteoblasts is significantly reduced by NF-{kappa}B and TLR4 inhibitors. Finally, RNA sequencing demonstrated that the deficits in load-induced bone formation in CKO mice can be attributed to dysregulated inflammatory signaling. In total, our study reveals a novel role for TLR4 in skeletal adaptation to mechanical loading in bone, which may enable new therapeutic strategies for diseases of low bone mass and provide new targets for musculoskeletal pain relief.

cell biology↗

bioRxiv2019

Multi-Differentiation Potential Is Necessary For Optimal Tenogenesis Of Tendon Stem Cells

BackgroundTendon injury is a significant clinical problem, and regenerative treatments are limited by our understanding of endogenous tendon stem cells. Recent evidence suggests that tendon stem cells are diverse in phenotypic character, and may in fact exist on a spectrum of differentiation capacities. However, the functional significance of each differentiation phenotype is poorly understood. Toward this end, we performed a comprehensive assessment of differentiation capacity toward four connective tissue lineages (adipose, bone, cartilage and tendon) with clonal tendon stem cell lines to: 1) evaluate the differences, if any, in tenogenic potential, and 2) identify the relationships in differentiation phenotype and proliferation capacity. MethodsTendon stem cells were derived from whole equine flexor tendons for this study (N=3). Clonal tendon stem lines were generated by low-density cell plating, and subjected to standard assays of tri-lineage differentiation and population doublings. For tenogenesis, a previously engineered three-dimensional hydrogel construct was incorporated. Differentiation was quantified by the relative gene expression of lineage-specific markers, and confirmed with lineage-specific cell staining. Tenogenesis was further analyzed by hydrogel contraction and histomorphometry. Statistical significance was determined using analysis of variance and post-hoc Tukeys tests. ResultsThree distinct tendon stem cell phenotypes were identified, namely differentiation toward: 1) adipose, bone, cartilage and tendon, 2) bone, cartilage and tendon, or 3) adipose, cartilage and tendon. Further, a positive correlation was found in the ability to differentiate toward all four lineages and the generation of a robust, composite tendon-like construct upon tenogenesis, manifested by the strongest expressions of scleraxis and mohawk, and parallel alignment of tenocyte-like cells with elongated cell morphologies. Significantly increased numbers of cumulative cell population doublings were seen in the absence of adipogenic potential in clonal tendon stem cell lines. ConclusionsOur study strengthens reports on the heterogeneous character of tendon stem cells and identifies key differences in their differentiation and proliferative potentials. Isolation of potent tendon stem cell populations from tendon stromal fractions may yield improved therapeutic benefits in clinical models of repair and promote a native, regenerative phenotype in engineered tendons. Future studies may be targeted to understanding the functional contributions of each tendon stem cell phenotype in vivo, and identifying additional cell phenotypes.

cell biology↗