Bao-Ting Zhang

A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy

Metabolic skeletal disorders associated with impaired bone formation are a major clinical challenge. One approach to treat these defects is to silence bone-formation–inhibitory genes by small interference RNAs (siRNAs) in osteogenic-lineage cells that occupy the niche surrounding the bone-formation surfaces. We developed a targeting system involving dioleoyl trimethylammonium propane (DOTAP)-based cationic liposomes attached to six repetitive sequences of aspartate, serine, serine ((AspSerSer)6) for delivering siRNAs specifically to bone-formation surfaces. Using this system, we encapsulated an osteogenic siRNA that targets casein kinase-2 interacting protein-1 (encoded by Plekho1, also known as Plekho1). In vivo systemic delivery of Plekho1 siRNA in rats using our system resulted in the selective enrichment of the siRNAs in osteogenic cells and the subsequent depletion of Plekho1. A bioimaging analysis further showed that this approach markedly promoted bone formation, enhanced the bone micro-architecture and increased the bone mass in both healthy and osteoporotic rats. These results indicate (AspSerSer)6-liposome as a promising targeted delivery system for RNA interference–based bone anabolic therapy.

Aptamer-functionalized lipid nanoparticles targeting osteoblasts as a novel RNA interference–based bone anabolic strategy

Currently, major concerns about the safety and efficacy of RNA interference (RNAi)-based bone anabolic strategies still exist because of the lack of direct osteoblast-specific delivery systems for osteogenic siRNAs. Here we screened the aptamer CH6 by cell-SELEX, specifically targeting both rat and human osteoblasts, and then we developed CH6 aptamer–functionalized lipid nanoparticles (LNPs) encapsulating osteogenic pleckstrin homology domain-containing family O member 1 (Plekho1) siRNA (CH6-LNPs-siRNA). Our results showed that CH6 facilitated in vitro osteoblast-selective uptake of Plekho1 siRNA, mainly via macropinocytosis, and boosted in vivo osteoblast-specific Plekho1 gene silencing, which promoted bone formation, improved bone microarchitecture, increased bone mass and enhanced mechanical properties in both osteopenic and healthy rodents. These results indicate that osteoblast-specific aptamer-functionalized LNPs could act as a new RNAi-based bone anabolic strategy, advancing the targeted delivery selectivity of osteogenic siRNAs from the tissue level to the cellular level.

Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation.

Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation.

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.

Electrical Stimulation Influences Satellite Cell Proliferation and Apoptosis in Unloading-Induced Muscle Atrophy in Mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.

Pathways of Ca2+ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle

Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.

Combination of inflammation-related cytokines promotes long-term muscle stem cell expansion.

Muscle stem cells (MuSCs, satellite cells) are the major contributor to muscle regeneration. Like most adult stem cells, long-term expansion of MuSCs in vitro is difficult. The in vivo muscle regeneration abilities of MuSCs are quickly lost after culturing in vitro, which prevents the potential applications of MuSCs in cell-based therapies. Here, we establish a system to serially expand MuSCs in vitro for over 20 passages by mimicking the endogenous microenvironment. We identified that the combination of four pro-inflammatory cytokines, IL-1α, IL-13, TNF-α, and IFN-γ, secreted by T cells was able to stimulate MuSC proliferation in vivo upon injury and promote serial expansion of MuSCs in vitro. The expanded MuSCs can replenish the endogenous stem cell pool and are capable of repairing multiple rounds of muscle injuries in vivo after a single transplantation. The establishment of the in vitro system provides us a powerful method to expand functional MuSCs to repair muscle injuries.

The beneficial effect of Icaritin on osteoporotic bone is dependent on the treatment initiation timing in adult ovariectomized rats

BACKGROUND Epimedium-derived flavonoids (EFs) have a potential to treat established osteoporosis in postmenopausal women. However, one of the main disadvantages of the compound is the high volume and dosage during long-term administration period. Meanwhile, the beneficial effect of EFs on osteoporotic bone depends greatly on the intervention timing. Whether icaritin (ICT), an active molecular compound from EFs, can exert beneficial effect on osteoporotic bone and whether the beneficial effect is also dependent on the intervention timing remain unknown. OBJECTIVE The objective of this study was to evaluate the effect of the early and late ICT treatment on bone turnover markers, trabecular architecture, bone remodeling, biomechanics, colony formation of bone marrow stromal cells and osteoblast, adipocyte and osteoclast-related gene expression in adult ovariectomized rats. METHODS Eighty 9-month-old female rats (n=8/group) were sham-operated (Sham) or ovariectomized (OVX). The OVX rats were subjected to ICT treatment initiation at 1 month (early treatment) and 3 months (late treatment) post-operation, respectively. The vehicle-treated Sham and OVX rats starting at month 1 and month 3 post-operation served as the corresponding controls (Sham and OVX controls) for early and late ICT treatment, respectively. Those Sham and OVX rats sacrificed immediately before early and late ICT treatment served as the pretreatment baseline controls. Both ICT and vehicle treatments lasted for 2 months. The bone turnover markers, trabecular architecture, bone remodeling and bone biomechanical properties were analyzed with biochemistry, microCT, histomorphometry and mechanical testing, respectively. The population of bone marrow stromal cells (BMSCs) and osteoblasts were evaluated with colony formation assays, respectively. The expression levels of osteoblast, adipocyte and osteoclast-related genes in bone marrow were assessed by real-time polymerase chain reaction (PCR), respectively. RESULTS At the tissue level, early ICT treatment remarkably restored the trabecular bone mass, trabecular architecture and bone biomechanical properties towards pretreatment Sham levels, and significantly increased bone formation from pretreatment OVX level and markedly inhibited bone resorption towards pretreatment Sham level, whereas late ICT treatment failed to have any effect. At the cellular and molecular level, early ICT treatment significantly increased the number of osteoblastic colonies and the level of osteoblast-related gene expression compared to pretreatment OVX levels and remarkably decreased adipocyte and osteoclast-related gene expression towards pretreatment Sham levels. Late ICT treatment failed to have beneficial effect on any of these parameters. CONCLUSION ICT can exert anabolic and anti-resorptive effect on osteoporotic bone. The beneficial effect of ICT treatment is dependent on the intervention timing in established osteoporosis induced by estrogen depletion.

The effects of low frequency electrical stimulation on satellite cell activity in rat skeletal muscle during hindlimb suspension.

BackgroundThe ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. It has been shown that chronic hindlimb unloading downregulates the satellite cell activity. This study investigated the role of low-frequency electrical stimulation on satellite cell activity during a 28 d hindlimb suspension in rats.ResultsMechanical unloading resulted in a 44% reduction in the myofiber cross-sectional area as well as a 29% and 34% reduction in the number of myonuclei and myonuclear domains, respectively, in the soleus muscles (P < 0.001 vs the weight-bearing control). The number of quiescent (M-cadherin+), proliferating (BrdU+ and myoD+), and differentiated (myogenin+) satellite cells was also reduced by 48-57% compared to the weight-bearing animals (P < 0.01 for all). Daily application of electrical stimulation (2 × 3 h at a 20 Hz frequency) partially attenuated the reduction of the fiber cross-sectional area, satellite cell activity, and myonuclear domain (P < 0.05 for all). Extensor digitorum longus muscles were not significantly altered by hindlimb unloading.ConclusionThis study shows that electrical stimulation partially attenuated the decrease in muscle size and satellite cells during hindlimb unloading. The causal relationship between satellite cell activation and electrical stimulation remain to be established.

Stretch-Induced Membrane Damage in Muscle: Comparison of Wild-Type and mdx Mice

One component of stretch-induced muscle damage is an increase in the permeability of the cell membrane. As a result soluble myoplasmic proteins leak out of the muscle into the plasma, extracellular proteins can enter the muscle, and extracellular ions, including calcium, are driven down their electrochemical gradient into the myoplasm. In Duchenne muscular dystrophy, caused by the absence of the cytoskeletal protein dystrophin, stretch-induced membrane damage is much more severe. The most popular theory to explain the occurrence of stretch-induced membrane damage is that stretched-contractions cause transient mechanically-induced defects in the membrane (tears or rips). Dystrophin, which is part of a mechanical link between the contractile machinery and the extracellular matrix, is thought to contribute to membrane strength so that in its absence mechanically-induced defects are worse. In our view the evidence that stretch-induced muscle damage causes increased membrane permeability is overwhelming but the evidence that the increased permeability is caused by mechanically-induced defects is weak. Instead we review the substantial evidence that the membrane permeability is a secondary consequence of the mechanical events in which elevated intracellular calcium and reactive oxygen species are important intermediaries.