Synthesis Studies and the Evaluation of C6 Raloxifene Derivatives.

Methodology is described for the synthesis of C6 derivatives of raloxifene, a prescribed drug for the treatment and prevention of osteoporosis. Studies have explored the incorporation of electron-withdrawing substituents at C6 of the benzothiophene core. Efficient processes are also examined to introduce hydrogen bond donor and acceptor functionality. Raloxifene derivatives are evaluated with in vitro testing to determine estrogen receptor (ER) binding affinity and gene expression in MC3T3 cells.

Effects of novel raloxifene analogs alone or in combination with mechanical loading in the Col1a2G610c/+ murine model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a hereditary bone disease in which gene mutations affect collagen formation, leading to a weak, brittle bone phenotype that can cause severe skeletal deformity and increased fracture risk. OI interventions typically repurpose osteoporosis medications to increase bone mass, but this approach does not address compromised tissue-level material properties. Raloxifene (RAL) is a mild anti-resorptive used to treat osteoporosis that has also been shown to increase bone strength by a-cellularly increasing bone bound water content, but RAL cannot be administered to children due to its hormonal activity. The goal of this study was to test a RAL analog with no estrogen receptor (ER) signaling but maintained ability to reduce fracture risk. The best performing analog from a previous analog characterization project, named RAL-ADM, was tested in an in vivo study. Female wildtype (WT) and Col1a2G610C/+ (G610C) mice were randomly assigned to treated or untreated groups, for a total of 4 groups (n = 15). Starting at 10 weeks of age, all mice underwent compressive tibial loading 3×/week to induce an anabolic bone formation response in conjunction with RAL-ADM treatment (0.5 mg/kg; 5×/week) for 6 weeks. Tibiae were scanned via microcomputed tomography then tested to failure in four-point bending. RAL-ADM had reduced ER affinity, and increased post-yield properties, but did not improve bone strength in OI animals, suggesting some properties can be improved by RAL analogs but further development is needed to create an analog with decidedly positive impacts to OI bone.

Changes in Bone Quality after Treatment with Etelcalcetide.

INTRODUCTION: Secondary hyperparathyroidism is associated with osteoporosis and fractures. Etelcalcetide is an intravenous calcimimetic for the control of hyperparathyroidism in patients on hemodialysis. Effects of etelcalcetide on the skeleton are unknown. METHODS: In a single-arm, open-label, 36-week prospective trial, we hypothesized that etelcalcetide improves bone quality and strength without damaging bone-tissue quality. Participants were 18 years or older, on hemodialysis ≥1 year, without calcimimetic exposure within 12 weeks of enrollment. We measured pretreatment and post-treatment areal bone mineral density by dual-energy X-ray absorptiometry, central skeleton trabecular microarchitecture by trabecular bone score, and peripheral skeleton volumetric bone density, geometry, microarchitecture, and estimated strength by high-resolution peripheral quantitative computed tomography. Bone-tissue quality was assessed using quadruple-label bone biopsy in a subset of patients. Paired t tests were used in our analysis. RESULTS: Twenty-two participants were enrolled; 13 completed follow-up (mean±SD age 51±14 years, 53% male, and 15% White). Five underwent bone biopsy (mean±SD age 52±16 years and 80% female). Over 36 weeks, parathyroid hormone levels declined 67%±9% ( P < 0.001); areal bone mineral density at the spine, femoral neck, and total hip increased 3%±1%, 7%±2%, and 3%±1%, respectively ( P < 0.05); spine trabecular bone score increased 10%±2% ( P < 0.001); and radius stiffness and failure load trended to a 7%±4% ( P = 0.05) and 6%±4% increase ( P = 0.06), respectively. Bone biopsy demonstrated a decreased bone formation rate (mean difference -25±4 µ m 3 / µ m 2 per year; P < 0.01). CONCLUSIONS: Treatment with etelcalcetide for 36 weeks was associated with improvements in central skeleton areal bone mineral density and trabecular quality and lowered bone turnover without affecting bone material properties. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: The Effect of Etelcalcetide on CKD-MBD (Parsabiv-MBD), NCT03960437.

Raloxifene Stimulates Estrogen Signaling to Protect Against Age- and Sex-Related Intervertebral Disc Degeneration in Mice.

Estrogen agonist raloxifene is an FDA-approved treatment of osteoporosis in postmenopausal women, which may also be a promising prophylactic for painful intervertebral disc (IVD) degeneration. Here, we hypothesized that 1) aging and biological sex contribute to IVD degeneration by reducing estrogen signaling and that 2) raloxifene stimulates estrogen signaling to protect against age- and sex-related IVD degeneration in mice. 2.5-month-old (male and female) and 22.5-month-old (female) C57Bl/6J mice were subcutaneously injected with raloxifene hydrochloride 5x/week for 6 weeks (n = 7-9/grp). Next, female mice were ovariectomized (OVX) or sham operated at 4 months of age and tissues harvested at 6 months (n = 5-6/grp). Advanced aging and OVX increased IVD degeneration score, weakened IVD strength, reduced estrogen receptor-α (ER-α) protein expression, and increased neurotransmitter substance P (SP) expression. Similar to aging and compared with male IVDs, female IVDs were more degenerated, mechanically less viscoelastic, and expressed less ER-α protein, but unlike the effect induced by aging or OVX, IVD mechanical force was greater in females than in males. Therapeutically, systemic injection of raloxifene promoted ER-α protein to quell these dysregulations by enlarging IVD height, alleviating IVD degeneration score, increasing the strength and viscoelastic properties of the IVD, and reducing IVD cell expression of SP in young-adult and old female mice. Transcriptionally, injection of raloxifene upregulated the gene expression of ER-α and extracellular matrix-related anabolism in young-adult and old IVD. In vertebra, advanced aging and OVX reduced trabecular BV/TV, whereas injection of raloxifene increased trabecular BV/TV in young-adult and old female mice, but not in young-adult male mice. In vertebra, advanced aging, OVX, and biological sex (females > males) increased the number of SP-expressing osteocytes, whereas injection of raloxifene reduced the number of SP-expressing osteocytes in young-adult female and male mice and old female mice. Overall, injection of estrogen agonist raloxifene in mice normalized dysregulation of IVD structure, IVD mechanics, and pain-related SP expression in IVD cells and osteocytes induced by aging and biological sex. These data suggest that, in addition to bone loss, raloxifene may relieve painful IVD degeneration in postmenopausal women induced by advanced age, biological sex, and estrogen depletion.

Segregating the effects of ferric citrate-mediated iron utilization and FGF23 in a mouse model of CKD.

Ferric citrate (FC) is an approved therapy for chronic kidney disease (CKD) patients as a phosphate (Pi) binder for dialysis-dependent CKD, and for iron deficiency anemia (IDA) in non-dialysis CKD. Elevated Pi and IDA both lead to increased FGF23, however, the roles of iron and FGF23 during CKD remain unclear. To this end, iron and Pi metabolism were tested in a mouse model of CKD (0.2% adenine) ± 0.5% FC for 6 weeks, with and without osteocyte deletion of Fgf23 (flox-Fgf23/Dmp1-Cre). Intact FGF23 (iFGF23) increased in all CKD mice but was lower in Cre+ mice with or without FC, thus the Dmp1-Cre effectively reduced FGF23. Cre+ mice fed AD-only had higher serum Pi than Cre- pre- and post-diet, and the Cre+ mice had higher BUN regardless of FC treatment. Total serum iron was higher in all mice receiving FC, and liver Tfrc, Bmp6, and hepcidin mRNAs were increased regardless of genotype; liver IL-6 showed decreased mRNA in FC-fed mice. The renal 1,25-dihydroxyvitamin D (1,25D) anabolic enzyme Cyp27b1 had higher mRNA and the catabolic Cyp24a1 showed lower mRNA in FC-fed mice. Finally, mice with loss of FGF23 had higher bone cortical porosity, whereas Raman spectroscopy showed no changes in matrix mineral parameters. Thus, FC- and FGF23-dependent and -independent actions were identified in CKD; loss of FGF23 was associated with higher serum Pi and BUN, demonstrating that FGF23 was protective of mineral metabolism. In contrast, FC maintained serum iron and corrected inflammation mediators, potentially providing ancillary benefit.

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) R47H Variant Causes Distinct Age- and Sex-Dependent Musculoskeletal Alterations in Mice.

Previous studies proposed the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor expressed in myeloid cells including microglia in brain and osteoclasts in bone, as a link between brain and bone disease. The TREM2 R47H variant is a known risk factor for Alzheimer's disease (AD), the most common form of dementia. To investigate whether altered TREM2 signaling could contribute to bone and skeletal muscle loss, independently of central nervous system defects, we used mice globally hemizygous for the TREM2 R47H variant (TREM2R47H/+ ), which do not exhibit AD pathology, and wild-type (WT) littermate control mice. Dxa/Piximus showed bone loss in female TREM2R47H/+ animals between 4 and 13 months of age and reduced cancellous and cortical bone (measured by micro-computed tomography [µCT]) at 13 months, which stalled out by 20 months of age. In addition, they exhibited decreased femoral biomechanical properties measured by three-point bending at 13 months of age, but not at 4 or 20 months. Male TREM2R47H/+ animals had decreased trabecular bone geometry but increased ultimate strain and failure force at 20 months of age versus WT. Only male TREM2R47H/+ osteoclasts differentiated more ex vivo after 7 days with receptor activator of nuclear factor κB ligand (RANKL)/macrophage colony-stimulating factor (M-CSF) compared to WT littermates. Yet, estrogen receptor alpha expression was higher in female and male TREM2R47H/+ osteoclasts compared to WT mice. However, female TREM2R47H/+ osteoclasts expressed less complement 3 (C3), an estrogen responsive element, and increased protein kinase B (Akt) activity, suggesting altered estrogen signaling in TREM2R47H/+ cells. Despite lower bone volume/strength in TREM2R47H/+ mice, skeletal muscle function measured by plantar flexion and muscle contractility was increased in 13-month-old female mutant mice. Overall, these data demonstrate that an AD-associated TREM2 variant can alter bone and skeletal muscle strength in a sex-dimorphic manner independent of central neuropathology, potentially mediated through changes in osteoclastic intracellular signaling. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Increased dosage and treatment time of Epigallocatechin-3-gallate (EGCG) negatively affects skeletal parameters in normal mice and Down syndrome mouse models.

Bone abnormalities affect all individuals with Down syndrome (DS) and are linked to abnormal expression of DYRK1A, a gene found in three copies in people with DS and Ts65Dn DS model mice. Previous work in Ts65Dn male mice demonstrated that both genetic normalization of Dyrk1a and treatment with ~9 mg/kg/day Epigallocatechin-3-gallate (EGCG), the main polyphenol found in green tea and putative DYRK1A inhibitor, improved some skeletal deficits. Because EGCG treatment improved mostly trabecular skeletal deficits, we hypothesized that increasing EGCG treatment dosage and length of administration would positively affect both trabecular and cortical bone in Ts65Dn mice. Treatment of individuals with DS with green tea extract (GTE) containing EGCG also showed some weight loss in individuals with DS, and we hypothesized that weights would be affected in Ts65Dn mice after EGCG treatment. Treatment with ~20 mg/kg/day EGCG for seven weeks showed no improvements in male Ts65Dn trabecular bone and only limited improvements in cortical measures. Comparing skeletal analyses after ~20mg/kg/day EGCG treatment with previously published treatments with ~9, 50, and 200 mg/kg/day EGCG showed that increased dosage and treatment time increased cortical structural deficits leading to weaker appendicular bones in male mice. Weight was not affected by treatment in mice, except for those given a high dose of EGCG by oral gavage. These data indicate that high doses of EGCG, similar to those reported in some treatment studies of DS and other disorders, may impair long bone structure and strength. Skeletal phenotypes should be monitored when high doses of EGCG are administered therapeutically.

Iron deficiency and high-intensity running interval training do not impact femoral or tibial bone in young female rats.

In the USA, as many as 20 % of recruits sustain stress fractures during basic training. In addition, approximately one-third of female recruits develop Fe deficiency upon completion of training. Fe is a cofactor in bone collagen formation and vitamin D activation, thus we hypothesised Fe deficiency may be contributing to altered bone microarchitecture and mechanics during 12-weeks of increased mechanical loading. Three-week old female Sprague Dawley rats were assigned to one of four groups: Fe-adequate sedentary, Fe-deficient sedentary, Fe-adequate exercise and Fe-deficient exercise. Exercise consisted of high-intensity treadmill running (54 min 3×/week). After 12-weeks, serum bone turnover markers, femoral geometry and microarchitecture, mechanical properties and fracture toughness and tibiae mineral composition and morphometry were measured. Fe deficiency increased the bone resorption markers C-terminal telopeptide type I collagen and tartate-resistant acid phosphatase 5b (TRAcP 5b). In exercised rats, Fe deficiency further increased bone TRAcP 5b, while in Fe-adequate rats, exercise increased the bone formation marker procollagen type I N-terminal propeptide. In the femur, exercise increased cortical thickness and maximum load. In the tibia, Fe deficiency increased the rate of bone formation, mineral apposition and Zn content. These data show that the femur and tibia structure and mechanical properties are not negatively impacted by Fe deficiency despite a decrease in tibiae Fe content and increase in serum bone resorption markers during 12-weeks of high-intensity running in young growing female rats.

Effects of Raloxifene and tibial loading on bone mass and mechanics in male and female mice.

Purpose: Raloxifene (RAL) is a selective estrogen receptor modulator (SERM) that has previously been shown to cause acellular benefits to bone tissue. Due to these improvements, RAL was combined with targeted tibial loading to assess if RAL treatment during periods of active bone formation would allow for further mechanical enhancements.Methods: Structural, mechanical, and microstructural effects were assessed in bone from C57BL/6 mice that were treated with RAL (0.5 mg/kg), tibial loading, or both for 6 weeks, beginning at 10 weeks of age.Results:Ex vivo microcomputed tomography (CT) images indicated RAL and loading work together to improve bone mass and architecture, especially within the cancellous region of males. Increases in cancellous bone volume fraction were heavily driven by increases in trabecular thickness, though there were some effects on trabecular spacing and number. In the cortical regions, RAL and loading both increased cross-sectional area, cortical area, and cortical thickness. Whole-bone mechanical testing primarily indicated the effects of loading. Further characterization through Raman spectroscopy and nanoindentation showed load-based changes in mineralization and micromechanics, while both loading and RAL caused changes in the secondary collagen structure. In contrast to males, in females, there were large load-based effects in the cancellous and cortical regions, resulting in increased whole-bone mechanical properties. RAL had less of an effect on cancellous and cortical architecture, though some effects were still present.Conclusion: RAL and loading work together to impact bone architecture and mechanical integrity, leading to greater improvements than either treatment individually.

Mechanical suppression of breast cancer cell invasion and paracrine signaling to osteoclasts requires nucleo-cytoskeletal connectivity.

Exercise benefits the musculoskeletal system and reduces the effects of cancer. The effects of exercise are multifactorial, where metabolic changes and tissue adaptation influence outcomes. Mechanical signals, a principal component of exercise, are anabolic to the musculoskeletal system and restrict cancer progression. We examined the mechanisms through which cancer cells sense and respond to low-magnitude mechanical signals introduced in the form of vibration. Low-magnitude, high-frequency vibration was applied to human breast cancer cells in the form of low-intensity vibration (LIV). LIV decreased matrix invasion and impaired secretion of osteolytic factors PTHLH, IL-11, and RANKL. Furthermore, paracrine signals from mechanically stimulated cancer cells, reduced osteoclast differentiation and resorptive capacity. Disconnecting the nucleus by knockdown of SUN1 and SUN2 impaired LIV-mediated suppression of invasion and osteolytic factor secretion. LIV increased cell stiffness; an effect dependent on the LINC complex. These data show that mechanical vibration reduces the metastatic potential of human breast cancer cells, where the nucleus serves as a mechanosensory apparatus to alter cell structure and intercellular signaling.

The loss of STAT3 in mature osteoclasts has detrimental effects on bone structure.

Signal Transducer and Activator of Transcription 3 (STAT3) has recently been shown to be involved in bone development and has been implicated in bone diseases, such as Job's Syndrome. Bone growth and changes have been known for many years to differ between sexes with male bones tending to have higher bone mass than female bones and older females tending to lose bone mass at faster rates than older males. Previous studies using conditional knock mice with Stat3 specifically deleted from the osteoblasts showed both sexes exhibited decreased bone mineral density (BMD) and strength. Using the Cre-Lox system with Cathepsin K promotor driving Cre to target the deletion of the Stat3 gene in mature osteoclasts (STAT3-cKO mice), we observed that 8-week old STAT3-cKO female femurs exhibited significantly lower BMD and bone mineral content (BMC) compared to littermate control (CN) females. There were no differences in BMD and BMC observed between male knock-out and male CN femurs. However, micro-computed tomography (µCT) analysis showed that both male and female STAT3-cKO mice had significant decreases in bone volume/tissue volume (BV/TV). Bone histomorphometry analysis of the distal femur, further revealed a decrease in bone formation rate and mineralizing surface/bone surface (MS/BS) with a significant decrease in osteoclast surface in female, but not male, STAT3-cKO mice. Profiling gene expression in an osteoclastic cell line with a knockdown of STAT3 showed an upregulation of a number of genes that are directly regulated by estrogen receptors. These data collectively suggest that regulation of STAT3 differs in male and female osteoclasts and that inactivation of STAT3 in osteoclasts affects bone turnover more in females than males, demonstrating the complicated nature of STAT3 signaling pathways in osteoclastogenesis. Drugs targeting the STAT3 pathway may be used for treatment of diseases such as Job's Syndrome and osteoporosis.

N-acetylcysteine (NAC), an anti-oxidant, does not improve bone mechanical properties in a rat model of progressive chronic kidney disease-mineral bone disorder.

Individuals with chronic kidney disease have elevated levels of oxidative stress and are at a significantly higher risk of skeletal fracture. Advanced glycation end products (AGEs), which accumulate in bone and compromise mechanical properties, are known to be driven in part by oxidative stress. The goal of this study was to study effects of N-acetylcysteine (NAC) on reducing oxidative stress and improving various bone parameters, most specifically mechanical properties, in an animal model of progressive CKD. Male Cy/+ (CKD) rats and unaffected littermates were untreated (controls) or treated with NAC (80 mg/kg, IP) from 30 to 35 weeks of age. Endpoint measures included serum biochemistries, assessments of systemic oxidative stress, bone morphology, and mechanical properties, and AGE levels in the bone. CKD rats had the expected phenotype that included low kidney function, elevated parathyroid hormone, higher cortical porosity, and compromised mechanical properties. NAC treatment had mixed effects on oxidative stress markers, significantly reducing TBARS (a measure of lipid peroxidation) while not affecting 8-OHdG (a marker of DNA oxidation) levels. AGE levels in the bone were elevated in CKD animals and were reduced with NAC although this did not translate to a benefit in bone mechanical properties. In conclusion, NAC failed to significantly improve bone architecture/geometry/mechanical properties in our rat model of progressive CKD.

6'-Methoxy Raloxifene-analog enhances mouse bone properties with reduced estrogen receptor binding.

Raloxifene (RAL) is an FDA-approved drug used to treat osteoporosis in postmenopausal women. RAL suppresses bone loss primarily through its role as a selective estrogen receptor modulator (SERM). This hormonal estrogen therapy promotes unintended side effects, such as hot flashes and increased thrombosis risk, and prevents the drug from being used in some patient populations at-risk for fracture, including children with bone disorders. It has recently been demonstrated that RAL can have significant positive effects on overall bone mechanical properties by binding to collagen and increasing bone tissue hydration in a cell-independent manner. A Raloxifene-Analog (RAL-A) was synthesized by replacing the 6-hydroxyl substituent with 6-methoxy in effort to reduce the compound's binding affinity for estrogen receptors (ER) while maintaining its collagen-binding ability. It was hypothesized that RAL-A would improve the mechanical integrity of bone in a manner similar to RAL, but with reduced estrogen receptor binding. Molecular assessment showed that while RAL-A did reduce ER binding, downstream ER signaling was not completely abolished. In-vitro, RAL-A performed similarly to RAL and had an identical concentration threshold on osteocyte cell proliferation, differentiation, and function. To assess treatment effect in-vivo, wildtype (WT) and heterozygous (OIM+/-) female mice from the Osteogenesis Imperfecta (OI) murine model were treated with either RAL or RAL-A from 8 weeks to 16 weeks of age. There was an untreated control group for each genotype as well. Bone microarchitecture was assessed using microCT, and mechanical behavior was assessed using 3-point bending. Results indicate that both compounds produced analogous gains in tibial trabecular and cortical microarchitecture. While WT mechanical properties were not drastically altered with either treatment, OIM+/- mechanical properties were significantly enhanced, most notably, in post-yield properties including bone toughness. This proof-of-concept study shows promising results and warrants the exploration of additional analog iterations to further reduce ER binding and improve fracture resistance.

The Importance of Biologically Active Vitamin D for Mineralization by Osteocytes After Parathyroidectomy for Renal Hyperparathyroidism.

Hypomineralized matrix is a factor determining bone mineral density. Increased perilacunar hypomineralized bone area is caused by reduced mineralization by osteocytes. The importance of vitamin D in the mineralization by osteocytes was investigated in hemodialysis patients who underwent total parathyroidectomy (PTX) with immediate autotransplantation of diffuse hyperplastic parathyroid tissue. No previous reports on this subject exist. The study was conducted in 19 patients with renal hyperparathyroidism treated with PTX. In 15 patients, the serum calcium levels were maintained by subsequent administration of alfacalcidol (2.0 µg/day), i.v. calcium gluconate, and oral calcium carbonate for 4 weeks after PTX (group I). This was followed in a subset of 4 patients in group I by a reduced dose of 0.5 µg/day until 1 year following PTX; this was defined as group II. In the remaining 4 patients, who were not in group I, the serum calcium (Ca) levels were maintained without subsequent administration of alfacalcidol (group III). Transiliac bone biopsy specimens were obtained in all groups before and 3 or 4 weeks after PTX to evaluate the change of the hypomineralized bone area. In addition, patients from group II underwent a third bone biopsy 1 year following PTX. A significant decrease of perilacunar hypomineralized bone area was observed 3 or 4 weeks after PTX in all group I and II patients. The area was increased again in the group II patients 1 year following PTX. In group III patients, an increase of the hypomineralized bone area was observed 4 weeks after PTX. The maintenance of a proper dose of vitamin D is necessary for mineralization by osteocytes, which is important to increase bone mineral density after PTX for renal hyperparathyroidism. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Voluntary Wheel Running Has Beneficial Effects in a Rat Model of CKD-Mineral Bone Disorder (CKD-MBD).

BACKGROUND: Reduced bone and muscle health in individuals with CKD contributes to their higher rates of morbidity and mortality. METHODS: We tested the hypothesis that voluntary wheel running would improve musculoskeletal health in a CKD rat model. Rats with spontaneous progressive cystic kidney disease (Cy/+ IU) and normal littermates (NL) were given access to a voluntary running wheel or standard cage conditions for 10 weeks starting at 25 weeks of age when the rats with kidney disease had reached stage 2-3 of CKD. We then measured the effects of wheel running on serum biochemistry, tissue weight, voluntary grip strength, maximal aerobic capacity (VO2max), body composition and bone micro-CT and mechanics. RESULTS: Wheel running improved serum biochemistry with decreased creatinine, phosphorous, and parathyroid hormone in the rats with CKD. It improved muscle strength, increased time-to-fatigue (for VO2max), reduced cortical porosity and improved bone microarchitecture. The CKD rats with voluntary wheel access also had reduced kidney cystic weight and reduced left ventricular mass index. CONCLUSIONS: Voluntary wheel running resulted in multiple beneficial systemic effects in rats with CKD and improved their physical function. Studies examining exercise interventions in patients with CKD are warranted.

Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality.

A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion: a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. The expression of matrix genes that contribute to bone material-level mechanical properties was elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.

Use of autologous adipose-derived mesenchymal stem cells for creation of laryngeal cartilage.

OBJECTIVES/HYPOTHESIS: Adipose-derived mesenchymal stem cells (ASCs) are an exciting potential cell source for tissue engineering because cells can be derived from the simple excision of autologous fat. This study introduces a novel approach for tissue-engineering cartilage from ASCs and a customized collagen oligomer solution, and demonstrates that the resultant cartilage can be used for laryngeal cartilage reconstruction in an animal model. STUDY DESIGN: Basic science experimental design. METHODS: ASCs were isolated from F344 rats, seeded in a customized collagen matrix, and cultured in chondrogenic differentiation medium for 1, 2, and 4 weeks until demonstrating cartilage-like characteristics in vitro. Large laryngeal cartilage defects were created in the F344 rat model, with the engineered cartilage used to replace the cartilage defects, and the rats followed for 1 to 3 months. Staining examined cellular morphology and cartilage-specific features. RESULTS: In vitro histological staining revealed rounded chondrocyte-appearing cells evenly residing throughout the customized collagen scaffold, with positive staining for cartilage-specific markers. The cartilage was used to successfully repair large cartilaginous defects in the rat model, with excellent functional results. CONCLUSIONS: This study is the first study to demonstrate, in an animal model, that ASCs cultured in a unique form of collagen oligomer can create functional cartilage-like grafts that can be successfully used for partial laryngeal cartilage replacement. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E123-E129, 2018.

Dairy Cows Naturally Infected with Bovine Leukemia Virus Exhibit Abnormal B- and T-Cell Phenotypes after Primary and Secondary Exposures to Keyhole Limpet Hemocyanin.

Bovine leukemia virus (BLV) is a retrovirus that is highly prevalent in US dairy herds: over 83% are BLV infected and the within-herd infection rate can be almost 50% on average. While BLV is known to cause lymphosarcomas, only 5% or fewer infected cattle will develop lymphoma; this low prevalence of cancer has historically not been a concern to dairy producers. However, more recent research has found that BLV+ cows without lymphoma produce less milk and have shorter lifespans than uninfected herdmates. It has been hypothesized that BLV infection interferes with normal immune function in infected cattle, and this could lead to reduced dairy production. To assess how naturally infected BLV+ cows responded to a primary and secondary immune challenge, 10 BLV+ and 10 BLV- cows were injected subcutaneously with keyhole limpet hemocyanin (KLH) and dimethyldioctadecylammonium bromide. B- and T-cell responses were characterized over the following 28 days. A total of 56 days after primary KLH exposure, cows were re-injected with KLH and B- and T-cell responses were characterized again over the following 28 days. BLV+ cows produced less KLH-specific IgM after primary immune stimulation; demonstrated fewer CD45R0+ B cells, altered proportions of CD5+ B cells, altered expression of CD5 on CD5+ B cells, and reduced MHCII surface expression on B cells ex vivo; exhibited reduced B-cell activation in vitro; and displayed an increase in BLV proviral load after KLH exposure. In addition, BLV+ cows had a reduced CD45R0+γδ+ T-cell population in the periphery and demonstrated a greater prevalence of IL4-producing T cells in vitro. All together, our results demonstrate that both B- and T-cell immunities are disrupted in BLV+ cows and that antigen-specific deficiencies can be detected in BLV+ cows even after a primary immune exposure.

Epigallocatechin-3-gallate (EGCG) consumption in the Ts65Dn model of Down syndrome fails to improve behavioral deficits and is detrimental to skeletal phenotypes.

Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in phenotypes including intellectual disability and skeletal deficits. Ts65Dn mice have three copies of ~50% of the genes homologous to Hsa21 and display phenotypes associated with DS, including cognitive deficits and skeletal abnormalities. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including neurological development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have previously shown that EGCG treatment (~10mg/kg/day) improves skeletal abnormalities in Ts65Dn mice, yet the same dose, as well as ~20mg/kg/day did not rescue deficits in the Morris water maze spatial learning task (MWM), novel object recognition (NOR) or balance beam task (BB). In contrast, a recent study reported that an EGCG-containing supplement with a dose of 2-3mg per day (~40-60mg/kg/day) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated if an EGCG dosage similar to that study would yield similar improvements in either cognitive or skeletal deficits. Ts65Dn mice and euploid littermates were given EGCG [0.4mg/mL] or a water control, with treatments yielding average daily intakes of ~50mg/kg/day EGCG, and tested on the multivariate concentric square field (MCSF)-which assesses activity, exploratory behavior, risk assessment, risk taking, and shelter seeking-and NOR, BB, and MWM. EGCG treatment failed to improve cognitive deficits; EGCG also produced several detrimental effects on skeleton in both genotypes. In a refined HPLC-based assay, its first application in Ts65Dn mice, EGCG treatment significantly reduced kinase activity in femora but not in the cerebral cortex, cerebellum, or hippocampus. Counter to expectation, 9-week-old Ts65Dn mice exhibited a decrease in Dyrk1a protein levels in Western blot analysis in the cerebellum. The lack of beneficial therapeutic behavioral effects and potentially detrimental skeletal effects of EGCG found in Ts65Dn mice emphasize the importance of identifying dosages of EGCG that reliably improve DS phenotypes and linking those effects to actions of EGCG (or EGCG-containing supplements) in specific targets in brain and bone.

Reduced humoral immunity and atypical cell-mediated immunity in response to vaccination in cows naturally infected with bovine leukemia virus.

Bovine leukemia virus (BLV) is a retrovirus that is widely distributed across US dairy herds: over 83% of herds are BLV-infected and within-herd infection rates can approach 50%. BLV infection reduces both animal longevity and milk production and can interfere with normal immune health. With such a high prevalence of BLV infection in dairy herds, it is essential to understand the circumstances by which BLV negatively affects the immune system of infected cattle. To address this question, BLV- and BLV+ adult, lactating Holstein dairy cows were vaccinated with Bovi-Shield GOLD® FP® 5 L5 HB and their immune response to vaccination was measured over the course of 28days. On day 0 prior to vaccination and days 7, 14 and 28 post-vaccination, fresh PBMCs were characterized for T and B cell ratios in the periphery. Plasma was collected to measure titers of IgM, IgG1 and IgG2 produced against bovine herpesvirus 1 (BHV1), Leptospira hardjo and L. pomona, as well as to characterize neutralizing antibody titers produced against BHV1 and bovine viral diarrhea virus types 1 and 2. On day 18 post-vaccination, PBMCs were cultured in the presence of BHV1 and flow cytometry was used to determine IFNγ production by CD4+, CD8+ and γδ T cells and to investigate CD25 and MHCII expression on B cells. BLV+ cows produced significantly lower titers of IgM against BHV1, L. hardjo and L. pomona and produced lower titers of IgG2 against BHV1. γδ T cells from BLV+ cows displayed a hyper reactive response to stimulation in vitro, although no differences were observed in CD4+ or CD8+ T cell activation. Finally, B cells from BLV+ cows exhibited higher CD25 expression and reduced MHCII expression in response to stimulation in vitro. All together, data from this study support the hypothesis that BLV+ cows fail to respond to vaccination as strongly as BLV- cows and, consequently, may have reduced protective immunity when compared to healthy BLV- cows.