A Novel EphA4 Signaling-Based Therapeutic Strategy for Osteoarthritis in Mice.

This study took advantage of the recent discovery that the EphA4 signaling has anti-catabolic effects on osteoclasts/macrophages/synoviocytes but pro-anabolic effects on articular chondrocytes and sought to develop an EphA4 signaling-based therapeutic strategy for osteoarthritis (OA) using a mouse model of OA/posttraumatic OA (PTOA). The injured joint of C57BL/6J mice received biweekly intraarticular injections of a soluble EphA4-binding ligand (EfnA4-fc) at 1 day after the tibial plateau injury or at 5 weeks post-injury. The animals were euthanized 5 weeks later. The injured right and contralateral uninjured left joints were analyzed for hallmarks of OA by histology. Relative severity was determined by a modified Mankin OA scoring system and serum COMP and CTX-II levels. Tibial plateau injury caused more severe OA in Epha4 null mice than in wild-type (WT) littermates, suggesting a protective role of EphA4 signaling in OA. A prototype strategy of an EphA4 signaling-based strategy involving biweekly injections of EfnA4-fc into injured joints was developed and was shown to be highly effective in preventing OA/PTOA when it was administered at 1 day post-injury and in treating OA/PTOA when it was applied after OA has been established. The efficacy of this prototype was dose- and time-dependent. The effects were not caused by the Fc moiety of EfnA4-fc. Other soluble EfnA ligands of EphA4, ie, EfnA1-fc and EfnA2-fc, were also effective. A prototype of a novel EphA4 signaling-based therapy was developed for OA/PTOA that not only reduces the progressive destruction of articular cartilage but may also promote regeneration of the damaged cartilage. © 2022 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

The EphA4 Signaling is Anti-catabolic in Synoviocytes but Pro-anabolic in Articular Chondrocytes.

The expression and activation of EphA4 in the various cell types in a knee joint was upregulated upon an intraarticular injury. To determine if EphA4 signaling plays a role in osteoarthritis, we determined whether deficient EphA4 expression (in EphA4 knockout mice) or upregulation of the EphA4 signaling (with the EfnA4-fc treatment) would alter cellular functions of synoviocytes and articular chondrocytes. In synoviocytes, deficient EphA4 expression enhanced, whereas activation of the EphA4 signaling reduced, expression and secretion of key inflammatory cytokines and matrix metalloproteases. Conversely, in articular chondrocytes, activation of the EphA4 signaling upregulated, while deficient EphA4 expression reduced, expression levels of chondrogenic genes (e.g., aggrecan, lubricin, type-2 collagen, and Sox9). EfnA4-fc treatment in wildtype, but not EphA4-deficient, articular chondrocytes promoted the formation and activity of acidic proteoglycan-producing colonies. Activation of the EphA4 signaling in articular chondrocytes upregulated Rac1/2 and downregulated RhoA via enhancing Vav1 and reducing Ephexin1 activation, respectively. However, activation of the EphA4 signaling in synoviocytes suppressed the Vav/Rac signaling while upregulated the Ephexin/Rho signaling. In summary, the EphA4 signaling in synoviocytes is largely of anti-catabolic nature through suppression of the expression of inflammatory cytokines and matrix proteases, but in articular chondrocytes the signaling is pro-anabolic in that it promotes the biosynthesis of articular cartilage. The contrasting action of the EphA4 signaling in synoviocytes as opposing to articular chondrocytes may in part be mediated through the opposite differential effects of the EphA4 signaling on the Vav/Rac signaling and Ephexin/Rho signaling in the two skeletal cell types.

Conditional Disruption of miR17~92 in Osteoclasts Led to Activation of Osteoclasts and Loss of Trabecular Bone In Part Through Suppression of the miR17-Mediated Downregulation of Protein-Tyrosine Phosphatase-oc in Mice.

This study sought to understand the regulation of an osteoclastic protein-tyrosine phosphatase (PTP-oc), a positive regulator of osteoclast activaty. Our past studies suggested that PTP-oc is regulated post-transcriptionally. The 3'-UTR of PTP-oc mRNA contains a target site for miR17. During osteoclastic differentiation, there was an inverse relationship between the cellular levels of miR17 (expressed as one of the six cluster genes of miR17~92) and PTP-oc mRNA. Overexpression of pre-miR17~92 in mouse osteoclast precursors reduced PTP-oc mRNA level and the size of the derived osteoclasts; whereas deletion of miR17~92 or inhibition of miR17 resulted in the formation of larger osteoclasts containing more nuclei that expressed higher PTP-oc mRNA levels and created larger resorption pits. Thus, PTP-oc-mediated osteoclast activation is modulated in part by miR17~92, particularly miR17. The miR17~92 osteoclast conditional knockout (cKO) mutants, generated by breeding miR17~92loxp/loxp mice with Ctsk-Cre mice, had lower Tb.BV/TV, Tb.BMD, Tb.Conn-Dens, Tb.N, and Tb.Th, but larger Tb.Sp, and greater bone resorption without a change in bone formation compared to littermate controls. The cKO marrow-derived osteoclasts were twice as large, contained twice as many nuclei, and produced twice as large resorption pits as osteoclasts of littermate controls. The expression of genes associated with osteoclast activation was increased in cKO osteoclasts, suggesting that deletion of miR17~92 in osteoclasts promotes osteoclast activation. The cKO osteoblasts did not show differences in cellular miR17 level, alkaline phosphatase activity, and bone nodule formation ability. In conclusion, miR17-92 negatively regulates the osteoclast activity, in part via the miR17-mediated suppression of PTP-oc in osteoclasts.

The effect of long-term hypoxia on tension and intracellular calcium responses following stimulation of the thromboxane A(2) receptor in the left anterior descending coronary artery of fetal sheep.

OBJECTIVE: The purpose of this study was to investigate the mechanisms of tension and intracellular calcium regulation following stimulation with the thromboxane A(2) receptor agonist U46619 in the left anterior descending coronary artery of fetal sheep exposed to long-term hypoxia. We hypothesized that there would be a reduction in intracellular calcium responses in long-term hypoxic left anterior descending coronary artery accompanied by an increase in calcium sensitivity of the contractile mechanism. METHODS: Pregnant sheep were kept at altitude (3820 m) from day 30 of gestation until day 140. Fetal hearts from long-term hypoxic and from a control, normoxic group were obtained and the left anterior descending coronary artery of the fetus was dissected, cleaned, and mounted in a bath (Jasco) in which tension and intracellular calcium [Ca(2+)](i), using Fura-2, could be measured simultaneously following stimulation of the thromboxane A(2) receptor with U46619. The role of intracellular calcium and the Rho kinase and protein kinase C pathways in the tension responses were investigated by maintaining intracellular calcium constant or by using the Rho kinase blocker, Y27632, or the protein kinase C blocker, GF109203-X. RESULTS: There was no difference in the tension dose-response to U46619 between the normoxic fetal and hypoxic fetal left anterior descending, although [Ca(2+)](i) was lower in the hypoxic fetal than normoxic fetal at the highest doses. When [Ca(2+)]( i) was maintained constant at baseline levels, U46619 produced the same tension dose-response in both normoxic fetal and hypoxic fetal left anterior descending as when [Ca(2+)](i) was allowed to rise. The tension response was abolished in both groups when the Rho kinase inhibitor, Y27632, was given either during or before stimulation with U46619. The protein kinase C blocker, GF109203-X, had no effect on the tension response in either group. CONCLUSIONS: Long-term hypoxia did not alter the tension response to thromboxane A(2) receptor stimulation in fetal left anterior descending. The contractions in response to U46619 were produced apparently completely by changes in calcium sensitivity through the Rho kinase pathway.

Effects of long-term, high-altitude hypoxia on tension and intracellular calcium responses in coronary arteries of fetal and adult sheep.

OBJECTIVES: We have previously shown that after exposure to long-term hypoxia, fetal coronary flow is maintained at control levels despite a 25% reduction in cardiac output. We also demonstrated that coronary vascular rings isolated from the long-term hypoxic fetuses and studied in well-oxygenated bath system displayed significantly reduced depolarization-induced contraction strength in response to KCl. To study the mechanism of reduced fetal coronary vascular responses to KCl-induced contractions following exposure to long-term hypoxia, we measured tension and intracellular calcium simultaneously, as well as L-type Ca2+ channel density and sensitivity. METHODS: Pregnant ewes were housed at altitude (3820 m) for approximately 110 days. At 138 to 141 days of gestation, long-term hypoxic and control animals were killed and fetal and adult left anterior descending coronary artery (LAD) was isolated and studied in a well-oxygenated bath system. Tension and intracellular calcium ([Ca2+]i) were measured simultaneously in response to increasing concentrations of KCl and, in addition, the sensitivity to the calcium channel blocker nifedipine was measured at a half maximal concentration of KCl. We also measured L-type Ca2+ channel density with (+)-[3H]PN200-110. RESULTS: L-type Ca2+ channel density was decreased by approximately 31% in the long-term hypoxic fetal, but not adult, LAD. Tension in the long-term hypoxic fetal and adult LAD was significantly lower at all concentrations of KCl. [Ca2+]i was lower at rest in both fetal and adult LAD from long-term hypoxic animals and increased to lower levels at all concentrations of KCl. The ratio of tension to [Ca2+]i was also lower at all concentrations of KCl. Sensitivity to nifedipine was unchanged. CONCLUSIONS: The reduced L-type Ca2+ channel density and the reduced [Ca2+]i response to KCl, as well as the reduced tension response to [Ca2+]i, could potentially be involved in the reduction in depolarization-induced contractions in LAD from long-term hypoxic fetuses. In hypoxic adults, reduced [Ca2+]i and reduced tension response to [Ca2+]i may be involved in the lower tension response to KCl-induced contractions.

Effects of long-term high-altitude hypoxia and troponin I phosphorylation on cardiac myofilament calcium responses in fetal and nonpregnant sheep.

OBJECTIVE: We studied the effects of long-term high-altitude hypoxia and protein kinase A (PKA) phosphorylation on calcium (Ca2+) responses of skinned cardiac papillary muscles from fetal and adult sheep. METHODS: Fetal and nonpregnant adult sheep were exposed to high-altitude (3820 m), long-term (approximately 110 days) hypoxia. Papillary muscles were isolated and mounted in well-oxygenated, temperature-controlled baths. After the papillary muscles were stimulated electrically to establish the diastolic tension that produced the maximum active contraction, the electrical stimulation was stopped, and the muscles were skinned with 1% vol/vol Triton-X-100. In protocol 1, the skinned muscles were exposed to activating solutions containing different calcium concentrations (pCa; from pCa 8.0 to pCa 4.0), which were prepared by varying the Ca-EGTA/EGTA ratio, and the steady-state tension was measured at each pCa. In protocol 2, the skinned muscles were contracted with activating solution containing a pCa of 5.0. After equilibration, the solution in some baths was changed to activating solution at the same pCa of 5.0 but also containing the catalytic subunit of PKA. The other baths were exchanged with activating solution at a pCa of 5.0 containing no PKA. We then measured the degree of tension reduction caused by PKA until tension reached a new steady state. RESULTS: In the long-term hypoxic fetal heart, the maximum tension response of right, but not left, ventricular skinned papillary muscle to Ca2+ was significantly less than that in control muscles. In the long-term hypoxic adult heart, the left ventricle, but not the right ventricle, displayed an increased maximum tension response to Ca2+ compared with control. Phosphorylation of troponin I (TnI) with PKA reduced active tension in both fetal ventricles of the long-term hypoxic group more than in hearts from control fetuses. In the adult, phosphorylation with PKA resulted in a larger decrease in tension in the left ventricle and a smaller decrease in tension in the right ventricle in the long-term hypoxic group, although the differences were small. CONCLUSION: In the long-term hypoxic fetal right ventricle, the decreased maximum tension response to Ca2+ is consistent with the decrease in myofibrillar magnesium-activated adenosine triphosphatase activity observed previously. The larger decrease in tension after PKA phosphorylation of TnI in the long-term hypoxic fetal left ventricle indicates a larger reduction in Ca2+ binding to troponin C.

Effects of long-term high-altitude hypoxia on myocardial protein kinase A activity and troponin I isoforms in fetal and nonpregnant sheep.

OBJECTIVE: In fetal sheep, we found that the augmentation of cardiac contractility by beta-adrenergic receptor (beta-AR) stimulation was reduced after exposure to long-term hypoxia. However, cyclic adenosine monophosphate (cAMP) production after beta-AR stimulation was higher in long-term hypoxic fetal sheep than in normoxic ones. Therefore, we studied the potential role of changes in myocardial protein kinase A (PKA) activity and troponin I (TnI) isoforms in fetal and nonpregnant sheep exposed to approximately 112 days of hypoxia at high altitude (3820 m). METHODS: Resting and maximally stimulated (by cAMP) PKA activity was measured by phosphorylation of the artificial peptide, Kemptide. Specificity was confirmed by inhibition with PKI, a specific PKA inhibitor. For TnI isoforms, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to resolve the proteins. We used monoclonal anti-cardiac TnI antibody (clone C5), which also cross-reacted with slow skeletal muscle TnI, to identify TnI isoforms. RESULTS: For the fetal hearts, resting PKA activity was significantly higher in the high-altitude group than the control group, but total PKA activity was not different between the normoxic and hypoxic groups. In the adult hearts, no significant difference was observed in either resting or total PKA activity between normoxic and hypoxic groups. For both the fetal and adult sheep, the predominant TnI was the cardiac isoform, and hypoxic exposure produced no change in the TnI isoform composition. CONCLUSIONS: Neither a reduction in PKA activity nor a change in TnI isoforms could explain the reduction in beta-receptor augmentation of cardiac contractility in fetal sheep exposed to long-term hypoxia.

Effects of long-term hypoxia and development on cardiac contractile proteins in fetal and adult sheep.

OBJECTIVE: We studied the effect of long-term, high-altitude hypoxia on cardiac myosin, actin, and troponin T (TnT) isoforms and Ca(2+)- and Mg(2+)-activated myofibrillar adenosine triphosphatase (ATPase) activities in fetal and adult sheep. METHODS: We exposed pregnant (beginning at day 30 of gestation) and nonpregnant sheep to high altitude (3820 m) for 110 days. Myosin, actin, and TnT isoforms were analyzed by Western analysis. In purified myofibrillar preparations, Ca(2+)(-) and Mg(2+)-ATPase activities were measured by the appearance of inorganic phosphate after the addition of NaATP and various concentrations of either calcium or magnesium to the reaction mixture. RESULTS: We found no change in myosin, actin, or TnT isoform composition after exposure to long-term hypoxia in either fetal or adult sheep. However, Mg(2+)-activated myofibrillar ATPase activity decreased significantly in the right ventricle of both fetus and adult after hypoxic exposure. There was also a significant maturational increase in both Ca(2+)- and Mg(2+)-ATPase activity in control animals. CONCLUSION: The decrease in Mg(2+)-activated myofibrillar ATPase activity might affect the decrease in cardiac contractility previously noted in the right ventricle of fetal sheep after exposure to long-term hypoxia. Likewise, the increase in Ca(2+)- and Mg(2+)-activated ATPase activities from the fetus to adult could partially explain the previously found maturational increase in cardiac contractility.