Nagat Frara

Osteoactivin promotes osteoblast adhesion through HSPG and αvβ1 integrin.

Osteoactivin (OA), also known as glycoprotein nmb (gpnmb) plays an important role in the regulation of osteoblast differentiation and function. OA induced osteoblast differentiation and function in vitro by stimulating alkaline phosphatase (ALP) activity, osteocalcin production, nodule formation, and matrix mineralization. Recent studies reported a role for OA in cell adhesion and integrin binding. In this study, we demonstrate that recombinant osteoactivin (rOA) as a matricellular protein stimulated adhesion, spreading and differentiation of MC3T3‐E1 osteoblast‐like cells through binding to αvβ1 integrin and heparan sulfated proteoglycans (HSPGs). MC3T3‐E1 cell adhesion to rOA was blocked by neutralizing anti‐OA or anti‐αv and β1 integrin antibodies. rOA stimulated‐osteoblast adhesion was also inhibited by soluble heparin and sodium chlorate. Interestingly, rOA stimulated‐osteoblast adhesion promoted an increase in FAK and ERK activation, resulting in the formation of focal adhesions, cell spreading and enhanced actin cytoskeleton organization. In addition, differentiation of primary osteoblasts was augmented on rOA coated‐wells marked by increased alkaline phosphatase staining and activity. Taken together, these data implicate OA as a matricellular protein that stimulates osteoblast adhesion through binding to αvβ1 integrin and cell surface HSPGs, resulting in increased cell spreading, actin reorganization, and osteoblast differentiation with emphasis on the positive role of OA in osteogenesis. J. Cell. Biochem. 115: 1243–1253, 2014.

Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In Vivo and Osteoprogenitor Differentiation Ex Vivo.

Initial identification of osteoactivin (OA)/glycoprotein non‐melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post‐fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV‐promoter (OA‐Tg). Western blot analysis showed increased OA/gpnmb in OA‐Tg osteoblasts, compared to wild‐type (WT). In OA‐Tg mouse femurs versus WT littermates, micro‐CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA‐Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over‐expressed in osteoclasts in OA‐Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX‐1 and RANK‐L, and decreased osteoclast numbers in OA‐Tg, compared to WT mice, indicating decreased bone remodeling in OA‐Tg mice. The proliferation rate of OA‐Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA‐Tg osteoprogenitors. Quantitative RT‐PCR analysis showed increased TGF‐β1 and TGF‐β receptors I and II expression in OA‐Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. J. Cell. Physiol. 230: 72–83, 2016.

Prolonged performance of a high repetition low force task induces bone adaptation in young adult rats, but loss in mature rats.

We have shown that prolonged repetitive reaching and grasping tasks lead to exposure-dependent changes in bone microarchitecture and inflammatory cytokines in young adult rats. Since aging mammals show increased tissue inflammatory cytokines, we sought here to determine if aging, combined with prolonged performance of a repetitive upper extremity task, enhances bone loss. We examined the radius, forearm flexor muscles, and serum from 16 mature (14-18 months of age) and 14 young adult (2.5-6.5 months of age) female rats after performance of a high repetition low force (HRLF) reaching and grasping task for 12 weeks. Young adult HRLF rats showed enhanced radial bone growth (e.g., increased trabecular bone volume, osteoblast numbers, bone formation rate, and mid-diaphyseal periosteal perimeter), compared to age-matched controls. Mature HRLF rats showed several indices of radial bone loss (e.g., decreased trabecular bone volume, and increased cortical bone thinning, porosity, resorptive spaces and woven bone formation), increased osteoclast numbers and inflammatory cytokines, compared to age-matched controls and young adult HRLF rats. Mature rats weighed more yet had lower maximum reflexive grip strength, than young adult rats, although each age group was able to pull at the required reach rate (4 reaches/min) and required submaximal pulling force (30 force-grams) for a food reward. Serum estrogen levels and flexor digitorum muscle size were similar in each age group. Thus, mature rats had increased bone degradative changes than in young adult rats performing the same repetitive task for 12 weeks, with increased inflammatory cytokine responses and osteoclast activity as possible causes.

Substance P increases CCN2 dependent on TGF-beta yet Collagen Type I via TGF-beta1 dependent and independent pathways in tenocytes

ABSTRACT Transforming growth factor beta 1 (TGFbeta-1) and connective tissue growth factor (CCN2) are important mediators of tissue repair and fibrosis, with CCN2 functioning as a downstream mediator of TGFβ-1. Substance P (SP) is also linked to collagen production in tenocytes. A link between SP, TGFbeta-1 and CCN2 has yet to be established in tenocytes or fibrogenic processes. We sought to determine whether SP induces tenocyte proliferation, CCN2, or collagen production via TGFbeta-1 signaling or independently in rat primary tenocytes. Tenocytes were isolated from rat tendons, cultured and stimulated by SP and/or TGFbeta-1. Cultured cells expressed proteins characteristic of tenocytes (vimentin and tenomodulin) and underwent increased proliferation dose dependently after SP and TGFbeta-1 treatments, alone or combined (more than SP alone when combined). SP induced TGFbeta-1 expression in tenocytes in both dose- and time-dependent manners. SP and TGFbeta-1, alone or combined, stimulated CCN2 expression in tenocytes and their supernatants after both 24 and 48 h of stimulation; a response blocked with addition of a TGFbeta-1 receptor inhibitor. In contrast, SP potentiated collagen type I secretion by tenocytes, a response abrogated by the TGFbeta-1 receptor inhibitor after 48 h of stimulation, but not after the shorter 24 h of stimulation. Our findings suggest that both SP and TGFbeta-1 can stimulate tenocyte fibrogenic processes, albeit differently. TGFbeta-1 pathway signaling was involved in CCN2 production at all time points examined, while SP induced collagen type I production independently prior to the onset of signaling through the TGFbeta-1 pathway.

Prolonged high force high repetition pulling induces trabecular bone loss and microcracks, while low force high repetition pulling induces bone anabolism

We have an operant rat model of upper extremity reaching and grasping in which we examined the impact of performing a high force high repetition (High-ForceHR) versus a low force low repetition (Low-ForceHR) task for 18 weeks on the radius, compared to age-matched controls. High-ForceHR rats performed at 4 reaches/min and 50% of their maximum voluntary pulling force for 2 hrs/day, 3 days/wk. Low-ForceHR rats performed at 6% maximum voluntary pulling force. High-ForceHR rats showed decreased trabecular bone volume in the distal radius and increased catabolic indices (microcracks and increased osteocyte apoptosis), compared to controls. In contrast, Low-ForceHR rats showed increased trabecular bone volume, and no microcracks or osteocyte apoptosis. Thus, prolonged performance of an upper extremity reaching and grasping task is loading-dependent with high force loading leading to accumulation of bone microdamage. The target grasp force (1.27N) of the High-ForceHR rats was 50% of their mean maximum voluntary force of 2.45N, easily below the predicted maximum acceptable force (MAF) of 1.64N (predicted from published equations). The presence of microcracks and bone loss leads us to suggest that the published equations over-estimated the MAF on these tissues by 1.3 fold.

Determining integrity of bladder innervation and smooth muscle function 1 year after lower spinal root transection in canines

To assess bladder smooth muscle function and innervation after long‐term lower spinal root transection in canines.

AB314. SPR-41 Localization of neuromuscular nicotinic receptors in the functionally reinnervated canine bladder after prolonged decentralization

Objective We previously found that intravenous succinylcholine, a depolarizing neuromuscular nicotinic receptors blocker, prevents bladder contractions induced by new neuronal pathways established by nerve transfer in decentralized dogs. We studied the detrusor pressure response in vivo and contractile response of bladder smooth muscle strips in vitro from sham, decentralized and reinnervated animals to localize the neuromuscular nicotinic receptors involved. Methods Three groups of female mongrel hound dogs were used: sham (N=4), 12-month decentralized (N=3) and 6-month reinnervated (N=3). Decentralization was created by bilateral transection of all spinal roots caudal to L7, including the dorsal roots of L7 and the hypogastric nerves. Reinnervation was created by bilateral transfer of the obturator nerve to anterior vesical branches of the pelvic nerve. Two-way ANOVAs and Sidak post-hoc tests were used to determine group differences. Results In reinnervated dogs, blockade of neuromuscular nicotinic receptor with intravenous injections of the competitive antagonist atracurium besylate significantly reduced the increase in detrusor pressure induced by electrical stimulation of the transferred obturator nerve (L1 or L2). Atracurium did not block the increase in pressure induced by stimulation of sacral nerve roots in sham-operated controls. In vitro, neither the competitive neuromuscular nicotinic receptor antagonist d-tubocurarine nor the ganglionic antagonist hexamethonium inhibited electric field stimulation (EFS)-induced contractions of reinnervated or sham-operated control bladder strips. No contractile response was elicited in the presence of 1 µM tetrodotoxin (TTX) across groups. Similarly, EFS-evoked contractions were strongly reduced by 10 µM alpha, beta-methylene ATP (α,β-mATP) and 1 µM atropine in all groups relative to the vehicle (water). Conclusions In vivo blockade of nerve-evoked bladder pressure by atracurium in the reinnervated, but not sham operated controls, suggests that neuromuscular nicotinic receptors become involved in bladder contractions induced by the new neuronal pathway. Because d-tubocurarine did not block in vitro contractions induced by EFS in the reinnervated bladders, the neuromuscular nicotinic receptors involved in the new neuronal pathway must not be located in the bladder muscle or intramural ganglia and therefore, are likely in preganglionic neurons. TTX blockade validates that EFS-induced contractions at all frequencies were nerve-evoked. Both muscarinic and purinergic components contributed similarly to neurotransmission based on response to blockade of nerve evoked muscle strip contractions with a combination of atropine and α,β-mATP. Funding Source(s) NIH-NINDS NS070267

AB301. SPR-28 Determining integrity of the nerve-smooth muscle functional unit of the bladder after long-term decentralization

Objective Somatic nerve transection causes rapid loss of skeletal muscle mass and contractility. While skeletal muscle degeneration following nerve injury has been well investigated, less is known about the effects of autonomic nerve transection on smooth muscle. We explored changes in the nerve-smooth muscle functional unit following sacral root decentralization to determine integrity after decreased innervation. Methods Female mixed-breed hound dogs were surgically decentralized by bilateral transection of all spinal roots caudal to L7, including the dorsal root of L7 in a subgroup. Three weeks prior to the terminal surgery, bladders were injected cystoscopically with fluorogold around the ureterovesical junction for retrograde neuronal labeling. Tissue function was tested during the terminal procedure after 6-month (n=2) and 12-month (n=6) decentralization and compared to sham/unoperated control animals (n=13). Immediately prior to euthanasia, in vivo detrusor pressure after stimulation of nerves originating from the pelvic plexus (e.g., the anterior vesicle branch) was recorded. Collected bladder and pelvic plexus tissues from controls and 6-month decentralized dogs were harvested (n=3–6/group), cryosectioned, and examined for fluorogold labeling. Bladder tissues were stained for caspase-3 and immunostaining was quantified. Gastric tissue and red blood cells within bladder walls were used as positive controls. Data was analyzed using unpaired ANOVA. Results Nerve stimulation caused a robust increase in detrusor pressure in both control and decentralized groups. Likewise, abundant fluorogold-labeled neuronal cell bodies were observed in ganglia in the pelvic plexus of both sham and decentralized animals. Immunohistochemical stain for caspase-3 showed no difference across groups. Also, we did not observe co-localization of fluorogold-positive neuronal tissue and caspase-3, or presence of caspase-3 in smooth muscle fibers of the bladder wall. Conclusions The presence of fluorogold-labeled pelvic plexus ganglia in decentralized animals demonstrates that the ganglia remained intact up to 6 months after decentralization. Caspase-3 staining results showed no increase in apoptosis in the neuronal tissues or bladder smooth muscle in decentralized dogs, suggesting no increased apoptotic cell death. No significant difference between detrusor pressure responses across groups after nerve-evoked stimulation indicates that the nerve-smooth muscle functional unit of the bladder is intact up to 12 months after injury and therefore, nerve reinnervation strategies could be successful. Funding Source(s) NIH-NINDS NS070267

AB318. SPR-45 Decentralization reduces nicotinic receptor-mediated canine bladder contractions in vitro

Objective Bladder function depends upon several complex signaling pathways that induce either contraction or relaxation. We performed nerve re-routing surgery on bladder of decentralized dogs (reinnervated) as a model with the goal of restoring bladder function in spinal cord injured patients. The neuromuscular nicotinic receptor blocker, succinylcholine blocks spinal root-stimulated bladder contraction in vivo in reinnervated dogs, but not in sham-operated dogs. Our lab explored the function and location of nicotinic receptors involved in bladder contraction, with emphasis on their possible role in the release of other neurotransmitters such as acetylcholine or ATP in sham-operated, 12-month-decentralized and immediate-reinnervated bladders. Methods Smooth muscle strips (mucosa denuded) were isolated from the region just rostral to the trigone and suspended in muscle baths. Strips were ranked based on their contractile response to a 3-min exposure to 120 mM KCl and sorted so that the average response in each group was equal. Strips were incubated with either 1 µM atropine (ATR), 1 µM tetrodotoxin (TTX), 10 µM alpha, beta-methylene ATP (α,β-ATP) or vehicle (water) and then induced to contract with the nicotinic receptor agonists 1,1 dimethyl-4-phenyl-piperazinium iodide (DMPP, 100 µM), TC2559 (100 µM) or epibatidine (10 µM) or 1 mM nicotine itself. Results The DMPP-induced contraction was not different between sham, reinnervated or decentralized bladders (11%, 3.3%, and 3.6% of KCl contraction respectively). While the epibatidine-induced contraction in shams was not different relative to that in the reinnervated (41% vs. 27% KCl respectively), it was significantly greater than that in decentralized bladders (13% of KCl). TC2559 did not induce bladder contractions. Nicotine-induced contractions in sham-operated controls were 16% of KCl. ATR completely blocked nicotine-induced contraction while α,β-ATP had no statistically significant effect in shams. TTX had no significant inhibitory effect on DMPP, epibatidine or nicotine-induced contraction in any group. Conclusions Nicotinic receptors mediate contraction in sham, reinnervated and decentralized bladders. This nicotinic receptor-mediated contraction is decreased after decentralization. TTX does not block nicotinic receptor-mediated contractions, indicating that action potentials are not required to induce contraction. In sham-operated dog bladders, the nicotine-induced contraction is blocked by ATR, suggesting that these nicotinic receptors are located on cholinergic nerve terminals and induce the release of acetylcholine, which activates muscarinic receptors on the smooth muscle. Funding Source(s) NIH-NINDS NS070267