Cyclooxygenase-2 plays a crucial role during myocardial patterning of developing chick.

Cyclooxygenase-2 (COX-2), a member of the Cyclooxygenase family, initiates the biosynthesis of prostanoids that regulates various cellular functions. Our pilot attempt revealed that the administration of etoricoxib, an inhibitor specific for COX-2, induces abnormal looping in the chicken heart. The present study attempts to reveal the mechanistic details of etoricoxib-induced abnormal cardiac looping. The activity of COX-2 was inhibited by administering 3.5 µg of etoricoxib into the egg's air cell on day zero of incubation. The gene and protein expression patterns of key mediators of heart development were then analyzed on day 2 (HH12) and day 3 (HH20). Reduced COX-2 activity altered the expressions of upstream regulators of organogenesis like Wnt11, BMP4, and SHH in the etoricoxib-exposed embryos. The observed expression shifts in the downstream regulators of myocardial patterning (MYOCD, HAND2, GATA4, GATA5, and GATA6) in the treated embryos corroborate the above results. In addition, the reduction in COX-2 activity hampered cardiomyocyte proliferation with a concomitant increase in the apoptosis rate. In conclusion, the collective effect of altered expression of signaling molecules of myocardial patterning and compromised cardiomyocyte turnover rate could be the reason behind the looping defects observed in the heart of etoricoxib-treated chick embryos.

Site-specific variation in the activity of COX-2 alters the pattern of wound healing in the tail and limb of northern house gecko by differentially regulating the expression of local inflammatory mediators.

The role of COX-2 induced PGE2 in the site-specific regulation of inflammatory mediators that facilitate disparate wound healing in the tail and limb of a lizard was studied by analysing their levels during various stages of healing. The activity of COX-2 and concentration of PGE2 surged during the early healing phase of tail along with the parallel rise in EP4 receptor. PGE2-EP4 interaction is corelated to early resolution (by 3 dpa) of inflammation by rising the antiinflammatory mediator IL-10. This likely causes reduction in proinflammatory mediators viz., iNOS, TNF-α, IL-6, IL-17 and IL-22. Conversely, in the limb, COX-2 derived PGE2 likely causes rise in inflammation through EP2 receptor-based signalling, as all the proinflammatory mediators stay elevated through the course of healing (till 9 dpa), while expression of IL-10 is reduced. This study brings to light the novel roles of IL-17 and IL-22 in programming wound healing. As IL-17 reduces in tail, IL-22 behaves in reparative way, causing conducive environment for scar-free wound healing. On the contrary, synergic elevation of both IL-17 and Il-22 form a micro-niche suitable for scarred wound healing in limb, thus obliterating its regenerative potential.

Inhibition of Cyclooxygenase-2 Alters Craniofacial Patterning during Early Embryonic Development of Chick.

A recent study from our lab revealed that the inhibition of cyclooxygenase-2 (COX-2) exclusively reduces the level of PGE2 (Prostaglandin E2) among prostanoids and hampers the normal development of several structures, strikingly the cranial vault, in chick embryos. In order to unearth the mechanism behind the deviant development of cranial features, the expression pattern of various factors that are known to influence cranial neural crest cell (CNCC) migration was checked in chick embryos after inhibiting COX-2 activity using etoricoxib. The compromised level of cell adhesion molecules and their upstream regulators, namely CDH1 (E-cadherin), CDH2 (N-cadherin), MSX1 (Msh homeobox 1), and TGF-ß (Transforming growth factor beta), observed in the etoricoxib-treated embryos indicate that COX-2, through its downstream effector PGE2, regulates the expression of these factors perhaps to aid the migration of CNCCs. The histological features and levels of FoxD3 (Forkhead box D3), as well as PCNA (Proliferating cell nuclear antigen), further consolidate the role of COX-2 in the migration and survival of CNCCs in developing embryos. The results of the current study indicate that COX-2 plays a pivotal role in orchestrating craniofacial structures perhaps by modulating CNCC proliferation and migration during the embryonic development of chicks.

New insights into the obligatory nature of cyclooxygenase-2 and PGE2 during early chick embryogenesis.

Temporal expression patterns and activity of two cyclooxygenase (COX-1 and COX-2) isoforms were analysed during early chick embryogenesis to evaluate their roles in development. COX-2 inhibition with etoricoxib resulted in significant structural anomalies such as anophthalmia (born without one or both eyes), phocomelia (underdeveloped or truncated limbs), and gastroschisis (an opening in the abdominal wall), indicating its significance in embryogenesis. Furthermore, the levels of PGE2, PGD2, PGF2α, and TXB2 were assessed using quantitative LC-MS/MS to identify which effector prostanoid (s) had their synthesis initiated by COX-2. COX-2 inhibition was only shown to reduce the level of PGE2 significantly, and hence it could be inferred that the later could be largely under the regulation of activated COX-2 in chick embryos. The compensatory increase in the activity of COX-1 observed in the etoricoxib-treated group helped to maintain the levels of PGD2, PGF2α, and TXB2. Though the roles of these three prostanoids in embryogenesis need to be further clarified, it appears that their contribution to the observed developmental anomalies is minimal. This study has shown that COX-2 is functionally active during chick embryogenesis, and it plays a central role in the structural configuration of several organs and tissues through its downstream effector molecule PGE2.

Early embryonic exposure to chlorpyrifos-cypermethrin combination induces pattern deficits in the heart of domestic hen.

Exposure to chlorpyrifos-cypermethrin combination during early development resulted in defective looping and ventricular noncompaction of heart in domestic chicken. The study was extended to elucidate the molecular basis of this novel observation. The primary culture of chicken embryonic heart cells showed a concentration-dependent loss of viability when challenged with this combination of technical-grade insecticides. Comet assay, DNA ladder assay, and analyses of appropriate markers at transcript and protein levels, revealed that chlorpyrifos-cypermethrin combination induced cell death by activating apoptosis. Parallelly, the tissues derived from control and experimental group hearts were checked for apoptotic markers, and the result was much similar to that of the in-vitro study. Further analysis showed that chlorpyrifos-cypermethrin combination deranged the expression pattern of the transcriptional regulators of cardiogenesis, namely TBX20, GATA5, HAND2, and MYOCD. This, together with heightened apoptosis, could well be the reason behind the observed structural anomalies in the heart of chlorpyrifos-cypermethrin poisoned embryos.