Gender affirming hormone therapy preserves skeletal maturation in young mice via the gut microbiome.

Gender affirming hormone therapy (GAHT) is often prescribed to transgender (TG) adolescents to alleviate gender dysphoria, but the impact of GAHT on the growing skeleton is unclear. We found GAHT to improve trabecular bone structure via increased bone formation in young male mice and not to affect trabecular structure in female mice. GAHT modified gut microbiome composition in both male and female mice. However, fecal microbiota transfers (FMT) revealed that GAHT-shaped gut microbiome was a communicable regulator of bone structure and turnover in male, but not in female mice. Mediation analysis identified two species of Bacteroides as significant contributors to the skeletal effects of GAHT in male mice, with Bacteroides supplementation phenocopying the effects of GAHT on bone. Bacteroides have the capacity to expand Treg populations in the gut. Accordingly, GAHT expanded intestinal regulatory T cells (Tregs) and stimulated their homing to the bone marrow (BM) in male but not in female mice. Attesting to the functional relevance of Tregs, pharmacological blockade of Treg expansion prevented GAHT-induced bone anabolism. In summary, in male mice GAHT stimulated bone formation and improved trabecular structure by promoting Treg expansion via a microbiome-mediated effect. In female mice GAHT neither improved nor impaired trabecular structure.

The Role of Osteogenic Effect and Vascular Function in Bone Health in Hypertensive Rats: A Study of Anti-hypertensive and Hemorheologic Drugs.

Vascular dysfunction contributes to the development of osteopenia in hypertensive patients, as decreased blood supply to bones results in tissue damage and dysfunction. The effect of anti-hypertensive medicines on bone mass in hypertensive individuals is inconclusive because of the varied mechanism of their action, and suggests that reducing blood pressure (BP) alone is insufficient to enhance bone mass in hypertension. Pentoxifylline (PTX), a hemorheological drug, improves blood flow by reducing blood viscosity and angiogenesis, also has an osteogenic effect. We hypothesized that improving vascular function is critical to increasing bone mass in hypertension. To test this, we screened various anti-hypertensive drugs for their in vitro osteogenic effect, from which timolol and hydralazine were selected. In adult female spontaneously hypertensive rats (SHRs), timolol and hydralazine did not improve vascular function and bone mass, but PTX improved both. In female SHR animals, PTX restored bone mass, strength and mineralization, up to the level of normotensive control rats. In addition, we observed lower blood vasculature in the femur of adult SHR animals, and PTX restored them. PTX also restored the bone vascular and angiogenesis parameters that had been impaired in OVX SHR compared to sham SHR. This study demonstrates the importance of vascular function in addition to increased bone mass for improving bone health as achieved by PTX without affecting BP, and suggests a promising treatment option for osteoporosis in hypertensive patients, particularly at-risk postmenopausal women.

TRPV4 regulates osteoblast differentiation and mitochondrial function that are relevant for channelopathy.

Different ion channels present in the osteoblast regulate the cellular functions including bio-mineralization, a process that is a highly stochastic event. Cellular events and molecular signaling involved in such process is poorly understood. Here we demonstrate that TRPV4, a mechanosensitive ion channel is endogenously present in an osteoblast cell line (MC3T3-E1) and in primary osteoblasts. Pharmacological activation of TRPV4 enhanced intracellular Ca2+-level, expression of osteoblast-specific genes and caused increased bio-mineralization. TRPV4 activation also affects mitochondrial Ca2+-levels and mitochondrial metabolisms. We further demonstrate that different point mutants of TRPV4 induce different mitochondrial morphology and have different levels of mitochondrial translocation, collectively suggesting that TRPV4-mutation-induced bone disorders and other channelopathies are mostly due to mitochondrial abnormalities. These findings may have broad biomedical implications.

Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice.

IL-17A (IL-17), a driver of the inflammatory phase of fracture repair, is produced locally by several cell lineages including γδ T cells and Th17 cells. However, the origin of these T cells and their relevance for fracture repair are unknown. Here, we show that fractures rapidly expanded callus γδ T cells, which led to increased gut permeability by promoting systemic inflammation. When the microbiota contained the Th17 cell-inducing taxon segmented filamentous bacteria (SFB), activation of γδ T cells was followed by expansion of intestinal Th17 cells, their migration to the callus, and improved fracture repair. Mechanistically, fractures increased the S1P receptor 1-mediated (S1PR1-mediated) egress of Th17 cells from the intestine and enhanced their homing to the callus through a CCL20-mediated mechanism. Fracture repair was impaired by deletion of γδ T cells, depletion of the microbiome by antibiotics (Abx), blockade of Th17 cell egress from the gut, or Ab neutralization of Th17 cell influx into the callus. These findings demonstrate the relevance of the microbiome and T cell trafficking for fracture repair. Modifications of microbiome composition via Th17 cell-inducing bacteriotherapy and avoidance of broad-spectrum Abx may represent novel therapeutic strategies to optimize fracture healing.

The microbiome restrains melanoma bone growth by promoting intestinal NK and Th1 cell homing to bone.

Bone metastases are frequent complications of malignant melanoma leading to reduced quality of life and significant morbidity. Regulation of immune cells by the gut microbiome influences cancer progression, but the role of the microbiome in tumor growth in bone is unknown. Using intracardiac or intratibial injections of B16-F10 melanoma cells into mice, we showed that gut microbiome depletion by broad-spectrum antibiotics accelerated intraosseous tumor growth and osteolysis. Microbiome depletion blunted melanoma-induced expansion of intestinal NK cells and Th1 cells and their migration from the gut to tumor-bearing bones. Demonstrating the functional relevance of immune cell trafficking from the gut to the bone marrow (BM) in bone metastasis, blockade of S1P-mediated intestinal egress of NK and Th1 cells, or inhibition of their CXCR3/CXCL9-mediated influx into the BM, prevented the expansion of BM NK and Th1 cells and accelerated tumor growth and osteolysis. Using a mouse model, this study revealed mechanisms of microbiota-mediated gut-bone crosstalk that are relevant to the immunological restraint of melanoma metastasis and tumor growth in bone. Microbiome modifications induced by antibiotics might have negative clinical consequences in patients with melanoma.

Oral Administration of Isovitexin, a Naturally Occurring Apigenin Derivative Showed Osteoanabolic Effect in Ovariectomized Mice: A Comparative Study with Teriparatide.

Isovitexin (apigenin-6C-glucopyranose) is found in several food items and medicinal plants. Recently, we showed that isovitexin stimulated osteoblast differentiation through mitochondrial biogenesis and respiration that required adiponectin receptors (AdipoRs). Here, we studied whether oral isovitexin has a bone anabolic effect in vivo. At first, using a femur osteotomy model in adult mice, we compared the bone regenerative effect of isovitexin and apigenin. Whereas isovitexin-stimulated bone formation at the osteotomy site at 2.5 mg/kg and 5 mg/kg dose, apigenin had no effect. Subsequently, we tested the effect of isovitexin (5 mg/kg) in ovariectomized (OVX) osteopenic mice and observed that it restored bone mass and architecture of trabecular bones (femur metaphysis and fifth lumbar vertebra/L5) and cortical bones (femur diaphysis). Isovitexin completely restored bone strength at L5 (compressive strength) and femur (bending strength) in OVX mice. The bone anabolic effect of isovitexin was demonstrated by the increased surface referent bone formation parameters, increased expression of osteogenic genes (Runx2, bone morphogenetic protein-2 and type 1 collagen) in bones, and increased serum procollagen type 1N-terminal propeptide in OVX mice and these were on a par with teriparatide. Isovitexin inhibited bone and serum sclerostin as well as the serum type I collagen cross-linked C-telopeptide in OVX mice. Isovitexin has an oral bioavailability of 14.58%. Taken together, our data show that isovitexin had a significant oral bioavailability that translated to osteoanabolic effect equivalent to teriparatide and inhibited bone resorption, which implied a durable effect over teriparatide.

Sirt3 ameliorates mitochondrial dysfunction and oxidative stress through regulating mitochondrial biogenesis and dynamics in cardiomyoblast.

Sirtuins are the endogenously present anti-aging protein deacetylases that regulate the mitochondrial biogenesis and function. Especially Sirt3, a mitochondrial sirtuin, is well known for maintaining mitochondrial function and health. In the present study, we have explored the novel role of Sirt3 in mitochondrial biogenesis and shown the role of Sirt3 in mito-nuclear communication through AMPK-α in Sirt3 knockdown and Sirt3 overexpressed H9c2 cells. The study found that impaired mitochondrial function in Sirt3-knockdown H9c2 cells was associated with decreased expression of mitochondrial DNA encoded genes, reduced SOD2 expression and activity. The study also revealed that Sirt3 knockdown affects mitochondrial biogenesis and dynamics. To further confirm the role of Sirt3 on mitochondrial biogenesis and health, we did Sirt3 overexpression in H9c2 cells. Sirt3 overexpression enhanced the expression of mitochondrial DNA encoded genes, increased SOD2 activity and altered mitochondrial dynamics. Sirt3 overexpression also caused an increase in mitochondrial biogenesis gene and protein (PGC-1α and TFAM) expression. All these changes were confirmed with mitochondrial functional parameters like basal respiration, maximal respiratory capacity, spare respiratory capacity and ATP production. We found decreased mitochondrial function in Sirt3-knockdown H9c2 cells when compared to control H9c2 cells. Together our data conclude that Sirt3 regulates cardiac mitochondrial health and function through the Sirt3-AMPKα-PGC-1α axis.

Diosmin, a citrus fruit-derived phlebotonic bioflavonoid protects rats from chronic kidney disease-induced loss of bone mass and strength without deteriorating the renal function.

Kidney Disease Improving Global Outcomes (KDIGO) 2017 Clinical Practice Guideline has recommended treatment decisions for patients with chronic kidney disease (CKD) with osteoporosis and/or high risk of fracture. Bisphosphonates, the first-line anti-osteoporosis drugs have the concern of worsening kidney functions. Moreover, despite impaired bone formation in CKD patients, teriparatide, the formation-stimulating drug is not recommended. Thus, there is an urgent need for safe and effective treatment of osteoporosis in CKD patients. Here, in CKD rats, we tested the osteoprotective effect of diosmin, a citrus-derived bioflavonoid used as a phlebotonic in chronic venous insufficiency and has a renoprotective effect. CKD was developed by 5/6th nephrectomy and diosmin at the human equivalent dose (100 mg kg-1) did not advance renal failure but reduced blood pressure to the level of sham control. Fibroblast growth factor-23 and parathyroid hormone were increased in CKD and diosmin suppressed both. CKD reduced bone mass and deteriorated the microarchitecture of trabecular bones, and diosmin maintained both to control levels. Bone formation and strength were impaired in the CKD and diosmin maintained these levels to control levels. Nanoindentation of bone showed that diosmin significantly increased tissue hardness over the control. Diosmetin, the metabolic surrogate of diosmin had comparable pharmacokinetic profiles between the control and CKD groups. Furthermore, diosmetin (50 mg kg-1) protected against CKD-induced bone loss. These data suggest that diosmin and its metabolic surrogate, diosmetin protect against CKD-induced osteopenia. Since diosmin has no renal adverse effect and protected bone mass and strength in CKD rats, we propose assessing its anti-osteoporosis effect in CKD patients.

Estradiol overcomes adiponectin-resistance in diabetic mice by regulating skeletal muscle adiponectin receptor 1 expression.

Adiponectin and insulin resistance creates a vicious cycle that exacerbates type 2 diabetes. Earlier, we observed that female leptin receptor-deficient BLKS mice (BKS-db/db) were more sensitive to an adiponectin mimetic GTDF than males, which led us to explore if E2 plays a crucial role in modulation of adiponectin-sensitivity. Male but not female BKS-db/db mice were resistant to metabolic effects of globular adiponectin treatment. Male BKS-db/db displayed reduced skeletal muscle AdipoR1 protein expression, which was consequent to elevated polypyrimidine tract binding protein 1 (PTB) and miR-221. E2 treatment in male BKS-db/db, and ovariectomized BALB/c mice rescued AdipoR1 protein expression via downregulation of PTB and miR-221, and also directly increased AdipoR1 mRNA by its classical nuclear receptors. Estrogen receptor regulation via dietary or pharmacological interventions may improve adiponectin resistance and consequently ameliorate insulin resistance in type 2 diabetes.

Adiponectin receptors by increasing mitochondrial biogenesis and respiration promote osteoblast differentiation: Discovery of isovitexin as a new class of small molecule adiponectin receptor modulator with potential osteoanabolic function.

Previously, we established adiponectin receptors (AdipoRs) as osteoanabolic target. To discover small molecule agonists of AdipoRs, we studied apigenin and apigenin-6C-glucopyranose (isovitexin) that induced osteoblast differentiation. In-silico, in vitro and omics-based studies were performed. Molecular docking using the crystal structures of AdipoRs showed different interaction profiles of isovitexin and apigenin. In osteoblasts, isovitexin but not apigenin rapidly phosphorylated AMP-activated protein kinase (pAMPK) which is downstream of AdipoRs and a master regulator of cellular energy metabolism, and upregulated expression of AdipoRs. Blocking AMPK abolished the osteogenic effect of isovitexin and its effect on AdipoR expression. Isovitexin upregulated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the mitochondrial biogenesis factor in osteoblasts, and the effect was blocked by AMPK inhibition. Upregulation of PGC-1α by isovitexin was accompanied by increased mitochondrial membrane proteins and mitochondrial DNA (mtDNA). Isovitexin via AdipoRs and PGC-1α induced oxidative phosphorylation (OxPhos) and ATP synthesis that resulted in osteoblast differentiation. Isovitexin had no agonistic/antagonistic activity and stimulatory/inhibitory effect in screening platforms for G protein-coupled receptors and kinases, respectively. In vivo, isovitexin upregulated AdipoRs and osteogenic genes, and increased mtDNA in rat calvarium. We conclude that isovitexin selectively via AdipoRs induced osteoblast differentiation that was fuelled by mitochondrial respiration.

Self-Assembling Nano-Globular Peptide from Human Lactoferrin Acts as a Systemic Enhancer of Bone Regeneration: A Novel Peptide for Orthopedic Application.

A technology for systemic and repeated administration of osteogenic factors for orthopedic use is an unmet medical need. Lactoferrin (∼80 kDa), present in milk, is known to support bone growth. We discovered a lactoferrin-mimetic peptide, LP2 (an 18-residue fragment from the N-terminus of the N-lobe of human lactoferrin), which self-assembles into a nano-globular assembly with a ß-sheet structure in an aqueous environment. LP2 is non-hemolytic and non-cytotoxic against human red blood cells and 3T3 fibroblasts, respectively, and appreciably stable in the human serum. LP2 through the bone morphogenetic protein-dependent mechanism stimulates osteoblast differentiation more potently than the full-length protein as well as the osteoblastic production of osteoprotegerin (an anti-osteoclastogenic factor). Consequently, daily subcutaneous administration of LP2 to rats and rabbits with osteotomy resulted in faster bone healing and stimulated bone formation in rats with a low bone mass more potently than that with teriparatide, the standard-of-care osteogenic peptide for osteoporosis. LP2 has skeletal bioavailability and is safe at the 15× osteogenic dose. Thus, LP2 is a novel peptide that can be administered systemically for the medical management of hard-to-heal fractures.

Therapeutic potential of phosphodiesterase inhibitors in the treatment of osteoporosis: Scopes for therapeutic repurposing and discovery of new oral osteoanabolic drugs.

Cyclic nucleotide phosphodiesterases (PDEs) are ubiquitously expressed enzymes that hydrolyze phosphodiester bond in the second messenger molecules including cAMP and cGMP. A wide range of drugs blocks one or more PDEs thereby preventing the inactivation of cAMP/cGMP. PDEs are differentially expressed in bone cells including osteoblasts, osteoclasts and chondrocytes. Intracellular increases in cAMP/cGMP levels in osteoblasts result in osteogenic response. Acting via the type 1 PTH receptor, teriparatide and abaloparatide increase intracellular cAMP and induce osteoanabolic effect, and many PDE inhibitors mimic this effect in preclinical studies. Since all osteoanabolic drugs are injectable and that oral drugs are considered to improve the treatment adherence and persistence, osteogenic PDE inhibitors could be a promising alternative to the currently available osteogenic therapies and directly assessed clinically in drug repurposing mode. Similar to teriparatide/abaloparatide, PDE inhibitors while stimulating osteoblast function also promote osteoclast function through stimulation of receptor activator of nuclear factor kappa-B ligand production from osteoblasts. In this review, we critically discussed the effects of PDE inhibitors in bone cells from cellular signalling to a variety of preclinical models that evaluated the bone formation mechanisms. We identified pentoxifylline (a non-selective PDE inhibitor) and rolipram (a PDE4 selective inhibitor) being the most studied inhibitors with osteogenic effect in preclinical models of bone loss at ≤ human equivalent doses, which suggest their potential for post-menopausal osteoporosis treatment through therapeutic repurposing. Subsequently, we treated pentoxifylline and rolipram as prototypical osteogenic PDEs to predict new chemotypes via the computer-aided design strategies for new drugs, based on the structural biology of PDEs.

Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF+ T cells and Th17 cells.

Estrogen deficiency causes a gut microbiome-dependent expansion of BM Th17 cells and TNF-α-producing T cells. The resulting increased BM levels of IL-17a (IL-17) and TNF stimulate RANKL expression and activity, causing bone loss. However, the origin of BM Th17 cells and TNF+ T cells is unknown. Here, we show that ovariectomy (ovx) expanded intestinal Th17 cells and TNF+ T cells, increased their S1P receptor 1-mediated (S1PR1-mediated) egress from the intestine, and enhanced their subsequent influx into the BM through CXCR3- and CCL20-mediated mechanisms. Demonstrating the functional relevance of T cell trafficking, blockade of Th17 cell and TNF+ T cell egress from the gut or their influx into the BM prevented ovx-induced bone loss. Therefore, intestinal T cells are a proximal target of sex steroid deficiency relevant for bone loss. Blockade of intestinal T cell migration may represent a therapeutic strategy for the treatment of postmenopausal bone loss.

TRPM8 channel inhibitor-encapsulated hydrogel as a tunable surface for bone tissue engineering.

A major limitation in the bio-medical sector is the availability of materials suitable for bone tissue engineering using stem cells and methodology converting the stochastic biological events towards definitive as well as efficient bio-mineralization. We show that osteoblasts and Bone Marrow-derived Mesenchymal Stem Cell Pools (BM-MSCP) express TRPM8, a Ca2+-ion channel critical for bone-mineralization. TRPM8 inhibition triggers up-regulation of key osteogenesis factors; and increases mineralization by osteoblasts. We utilized CMT:HEMA, a carbohydrate polymer-based hydrogel that has nanofiber-like structure suitable for optimum delivery of TRPM8-specific activators or inhibitors. This hydrogel is ideal for proper adhesion, growth, and differentiation of osteoblast cell lines, primary osteoblasts, and BM-MSCP. CMT:HEMA coated with AMTB (TRPM8 inhibitor) induces differentiation of BM-MSCP into osteoblasts and subsequent mineralization in a dose-dependent manner. Prolonged and optimum inhibition of TRPM8 by AMTB released from the gels results in upregulation of osteogenic markers. We propose that AMTB-coated CMT:HEMA can be used as a tunable surface for bone tissue engineering. These findings may have broad implications in different bio-medical sectors.

The gut microbiota is a transmissible determinant of skeletal maturation.

Genetic factors account for the majority of the variance of human bone mass, but the contribution of non-genetic factors remains largely unknown. By utilizing maternal/offspring transmission, cohabitation, or fecal material transplantation (FMT) studies, we investigated the influence of the gut microbiome on skeletal maturation. We show that the gut microbiome is a communicable regulator of bone structure and turnover in mice. In addition, we found that the acquisition of a specific bacterial strain, segmented filamentous bacteria (SFB), a gut microbe that induces intestinal Th17 cell expansion, was sufficient to negatively impact skeletal maturation. These findings have significant translational implications, as the identification of methods or timing of microbiome transfer may lead to the development of bacteriotherapeutic interventions to optimize skeletal maturation in humans. Moreover, the transfer of SFB-like microbes capable of triggering the expansion of human Th17 cells during therapeutic FMT procedures could lead to significant bone loss in fecal material recipients.

A prebiotic, short-chain fructo-oligosaccharides promotes peak bone mass and maintains bone mass in ovariectomized rats by an osteogenic mechanism.

In preclinical studies, fructooligosaccharide (FOS) showed beneficial skeletal effects but its effect on peak bone mass (PBM) and bone loss caused by estrogen (E2) deficiency has not been studied, and we set out to study these effects in rats. Short-chain (sc)-FOS had no effect on body weight, body composition, and energy metabolism of ovary intact (sham) and ovariectomized (OVX) rats. scFOS did not affect serum and urinary calcium and phosphorus levels, and on calcium absorption, although an increasing trend was noted in the sham group. Sham and OVX rats given scFOS had better skeletal parameters than their respective controls. scFOS treatment resulted in a higher bone anabolic response but had no effect on the catabolic parameters. scFOS increased serum levels of a short-chain fatty acid, butyrate which is known to have osteogenic effect. Our study for the first time demonstrates that in rats scFOS at the human equivalent dose enhances PBM and protects against E2 deficiency-induced bone loss by selective enhancement of new bone formation, and implicates butyrate in this process.

Calcium repletion to rats with calcipenic rickets fails to recover bone quality: A calcipenic "memory".

Calcipenic rickets is prevalent in underprivileged children in developing countries. Calcipenic rickets resulting from dietary calcium (Ca) deficiency decreases bone mass and deteriorates bone microstructure in humans. The effect of dietary Ca replenishment (CaR) on rachitic bones in animal models depends on the amount, critical period and duration of replenishment, however, the extent of recovery in various bone parameters including bone quality remains unclear. We investigated the effect of CaR in rat skeleton after inducing calcipenic rickets. Female SD rats (postnatal 28 days/P28) were rendered calcipenic by feeding calcium deficient (CaD) diet (0.1% Ca) till P70 while control SD rats were fed Ca sufficient diet (0.8% Ca). At P70, calcipenic rats were switched to 0.8% Ca diet till P150 for one group and P210 for another group (endpoint). The CaD groups received 0.1% Ca diet throughout the study (P210). In the CaD groups, serum Ca and phosphate, and bone mineral density (BMD) were significantly decreased whereas serum alkaline phosphatase (ALP), iPTH and CTX-1 were increased compared to age-matched controls. Moreover, at the endpoint, the CaD group had reduced bone mass, surface referent bone formation parameters, tissue mineralization and strength accompanied by the increased osteoid thickness and microarchitectural decay (measured by trabecular geometric parameters) with poor crystal packing. The CaR group showed complete recovery in serum Ca, iPTH, ALP and CTX-1, and BMD, however, the bone quality parameters including bone strength, microarchitectural decay, tissue mineralization, and crystallinity were incompletely restored. Decreased surface referent bone formation and increased unmineralized bones (osteoid) indicative of osteomalacia were also observed in the CaR group at P210 compared with control despite prolonged replenishment. We conclude that a prolonged Ca repletion following the induction of calcipenic rickets in rats although shows the recovery of biochemical measures of bone metabolism and bone mass, however, the bone quality remains compromised. This suggests that a "memory" of calcipenia occurring at the early growth stage persists in the skeleton of adult rats despite a prolonged Ca replenishment.

Selective dietary polyphenols induce differentiation of human osteoblasts by adiponectin receptor 1-mediated reprogramming of mitochondrial energy metabolism.

Anabolic therapies for osteoporosis including dietary polyphenols promote osteoblast function by influencing its energy metabolism. Among the dietary polyphenols, the beneficial skeletal effects of genistein (an isoflavone), kaempferol (a flavone), resveratrol (RES, a stilbenoid) and epigallocatechin gallate (EGCG, a catechin) have been reported in preclinical studies. We studied the action mechanism of these nutraceuticals on osteoblast bioenergetics. All stimulated differentiation of human fetal osteoblasts (hFOB). However, only EGCG and RES stimulated mitochondrial parameters including basal and maximum respiration, spare respiratory capacity and ATP production (a measure of the activity of electron transport chain/ETC). Increases in these parameters were due to increased mitochondrial biogenesis and consequent upregulation of several mitochondrial proteins including those involved in ETC. Rotenone blocked the osteogenic effect of EGCG and RES suggesting the mediatory action of mitochondria. Both compounds rapidly activated AMPK, and dorsomorphin (an AMPK inhibitor) abolished ATP production stimulated by these compounds. Moreover, EGCG and RES upregulated the mitochondrial biogenesis factor, PGC-1α which is downstream of AMPK activation, and silencing PGC-1α blocked their stimulatory effects on ATP production and hFOB differentiation. Adiponectin receptor 1 (AdipoR1) is an upstream regulator of PGC-1α, and both compounds increased the expression of AdipoR1 but not AdipoR2. Silencing AdipoR1 blocked the upregulation of EGCG/RES-induced PGC-1α and hFOB differentiation. In rat calvarium, both compounds increased AdipoR1, PGC-1α, and RunX2 (the osteoblast transcription factor) with a concomitant increase in mitochondrial copy number and ATP levels. We conclude that EGCG and RES display osteogenic effects by reprogramming osteoblastic bioenergetics by acting as the AdipoR1 agonists.

Skeletal restoration by phosphodiesterase 5 inhibitors in osteopenic mice: Evidence of osteoanabolic and osteoangiogenic effects of the drugs.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides and thereby regulate diverse cellular functions. The reports on the skeletal effects of PDE inhibitors are conflicting. Here, we screened 17 clinically used non-xanthine PDE inhibitors (selective and non-selective) using mouse calvarial osteoblasts (MCO) where the readout was osteoblast differentiation. From this screen, we identified sildenafil and vardenafil (both PDE5 inhibitors) having the least osteogenic EC50. Both drugs significantly increased vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expressions in MCO and the nitric oxide synthase inhibitor L-NAME completely blocked VEGF expression induced by these drugs. Sunitinib, a tyrosine receptor kinase inhibitor that also blocks VEGFR2 blocked sildenafil-/vardenafil-induced osteoblast differentiation. At half of their human equivalent doses, i.e. 6.0 mg/kg sildenafil and 2.5 mg/kg vardenafil, the maximum bone marrow level of sildenafil was 32% and vardenafil was 21% of their blood levels. At these doses, both drugs enhanced bone regeneration at the femur osteotomy site and completely restored bone mass, microarchitecture, and strength in OVX mice. Furthermore, both drugs increased surface referent bone formation and serum bone formation marker (P1NP) without affecting the resorption marker (CTX-1). Both drugs increased the expression of VEGF and VEGFR2 in bones and osteoblasts and increased skeletal vascularity. Sunitinib completely blocked the bone restorative and vascular effects of sildenafil and vardenafil in OVX mice. Taken together, our study suggested that sildenafil and vardenafil at half of their adult human doses completely reversed osteopenia in OVX mice by an osteogenic mechanism that was associated with enhanced skeletal vascularity.