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jcrmhscasereports · 1 year

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Retinal and choroidal vascular drop out in a case of severe phenotype of Flammer Syndrome. Rescue of the ischemic-preconditioning mimicking action of endogenous Erythropoietin (EPO) by off-label intra vitreal injection of recombinant human EPO (rhEPO) by Claude Boscher in Journal of Clinical Case Reports Medical Images and Health Sciences

Journal of Clinical Case Reports Medical Images and Health Sciences

ABSTRACT

Background: Erythropoietin (EPO) is a pleiotropic anti-apoptotic, neurotrophic, anti-inflammatory, and pro-angiogenic endogenous agent, in addition to its effect on erythropoiesis. Exogenous EPO is currently used notably in human spinal cord trauma, and pilot studies in ocular diseases have been reported. Its action has been shown in all (neurons, glia, retinal pigment epithelium, and endothelial) retinal cells. Patients affected by the Flammer Syndrome (FS) (secondary to Endothelin (ET)-related endothelial dysfunction) are exposed to ischemic accidents in the microcirculation, notably the retina and optic nerve.

Case Presentation: A 54 years old female patient with a diagnosis of venous occlusion OR since three weeks presented on March 3, 2019. A severe Flammer phenotype and underlying non arteritic ischemic optic neuropathy; retinal and choroidal drop-out were obviated. Investigation and follow-up were performed for 36 months with Retinal Multimodal Imaging (Visual field, SD-OCT, OCT- Angiography, Indo Cyanin Green Cine-Video Angiography). Recombinant human EPO (rhEPO)(EPREX®)(2000 units, 0.05 cc) off-label intravitreal injection was performed twice at one month interval. Visual acuity rapidly improved from 20/200 to 20/63 with disparition of the initial altitudinal scotoma after the first rhEPO injection, to 20/40 after the second injection, and gradually up to 20/32, by month 5 to month 36. Secondary cystoid macular edema developed ten days after the first injection, that was not treated via anti-VEGF therapy, and resolved after the second rhEPO injection. PR1 layer integrity, as well as protective macular gliosis were fully restored. Some level of ischemia persisted in the deep capillary plexus and at the optic disc.

Conclusion: Patients with FS are submitted to chronic ischemia and paroxystic ischemia/reperfusion injury that drive survival physiological adaptations via the hypoxic-preconditioning mimicking effect of endogenous EPO, that becomes overwhelmed in case of acute hypoxic stress threshold above resilience limits. Intra vitreal exogenous rhEPO injection restores retinal hypoxic-preconditioning adaptation capacity, provided it is timely administrated. Intra vitreal rhEPO might be beneficial in other retinal diseases of ischemic and inflammatory nature.

Key words : Erythropoietin, retinal vein occlusion, anterior ischemic optic neuropathy, Flammer syndrome, Primary Vascular Dysfunction, anti-VEGF therapy, Endothelin, microcirculation, off-label therapy.

INTRODUCTION

Retinal Venous Occlusion (RVO) treatment still carries insufficiencies and contradictions (1) due to the incomplete deciphering of the pathophysiology and of its complex multifactorial nature, with overlooking of factors other than VEGF up-regulation, notably the roles of retinal venous tone and Endothelin-1 (ET) (2-5), and of endothelial caspase-9 activation (6). Flammer Syndrome (FS)( (Primary Vascular Dysfunction) is related to a non atherosclerotic ET-related endothelial dysfunction in a context of frequent hypotension and increased oxidative stress (OS), that alienates organs perfusion, with notably changeable functional altered regulation of blood flow (7-9), but the pathophysiology remains uncompletely elucidated (8). FS is more frequent in females, and does not seem to be expressed among outdoors workers, implying an influence of sex hormons and light (7)(9). ET is the most potent pro-proliferative, pro-fibrotic, pro-oxidative and pro-inflammatory vasoconstrictor, currently considered involved in many diseases other than cardio-vascular ones, and is notably an inducer of neuronal apoptosis (10). It is produced by endothelial (EC), smooth vascular muscles (SVMC) and kidney medullar cells, and binds the surface Receptors ET-A on SVMC and ET-B on EC, in an autocrine and paracrine fashion. Schematically, binding on SVMC Receptors (i.e. through local diffusion in fenestrated capillaries or dysfunctioning EC) and on EC ones (i.e. by circulating ET) induce respectively arterial and venous vasoconstriction, and vasodilation, the latter via Nitrite oxide (NO) synthesis. ET production is stimulated notably by Angiotensin 2, insulin, cortisol, hypoxia, and antagonized by endothelial gaseous NO, itself induced by flow shear stress. Schematically but not exclusively, vascular tone is maintained by a complex regulation of ET-NO balance (8) (10-11). Both decrease of NO and increase of ET production are both a cause and consequence of inflammation, OS and endothelial dysfunction, that accordingly favour vasoconstriction; in addition ET competes for L-arginine substrate with NO synthase, thereby reducing NO bioavailability, a mechanism obviated notably in carotid plaques and amaurosis fugax (reviewed in 11).

Severe FS phenotypes are rare. Within the eye, circulating ET reaches retinal VSMC in case of Blood-Retinal-Barrier (BRB) rupture and diffuses freely via the fenestrated choroidal circulation, notably around the optic nerve (ON) head behind the lamina cribrosa, and may induce all pathologies related to acute ocular blood flow decrease (2-3)(5)(7-9). We previously reported two severe cases with rapid onset of monocular cecity and low vision, of respectively RVO in altitude and non arteritic ischemic optic neuropathy (NAION) (Boscher et al, Société Francaise d'Ophtalmologie and Retina Society, 2015 annual meetings).

Exogenous Recombinant human EPO (rhEPO) has been shown effective in humans for spinal cord injury (12), neurodegenerative and chronic kidney diseases (CKD) (reviewed in 13). Endogenous EPO is released physiologically in the circulation by the kidney and liver; it may be secreted in addition by all cells in response to hypoxic stress, and it is the prevailing pathway induced via genes up-regulation by the transcription factor Hypoxia Inducible Factor 1 alpha, among angiogenesis (VEGF pathway), vasomotor regulation (inducible NO synthase), antioxidation, and energy metabolism (14). EPO Receptor signaling induces cell proliferation, survival and differentiation (reviewed in 13), and targets multiple non hematopoietic pathways as well as the long-known effect on erythropoiesis (reviewed in 15). Of particular interest here, are its synergistic anti-inflammatory, neural antiapoptotic (16) pro-survival and pro-regenerative (17) actions upon hypoxic injury, that were long-suggested to be also indirect, via blockade of ET release by astrocytes, and assimilated to ET-A blockers action (18). Quite interestingly, endogenous EPO’s pleiotropic effects were long-summarized (back to 2002), as “mimicking hypoxic-preconditioning” by Dawson (19), a concept applied to the retina (20). EPO Receptors are present in all retinal cells and their rescue activation targets all retinal cells, i.e. retinal EC, neurons (photoreceptors (PR), ganglion (RGG) and bipolar cells), retinal pigment epithelium (RPE) osmotic function through restoration of the BRB, and glial cells (reviewed in 21), and the optic nerve (reviewed in 22). RhEPO has been tested experimentally in animal models of glaucoma, retinal ischemia-reperfusion (I/R) and light phototoxicity, via multiple routes (systemic, subconjunctival, retrobulbar and intravitreal injection (IVI) (reviewed in 23), and used successfully via IVI in human pilot studies, notably first in diabetic macular edema (24) (reviewed in 25 and 26). It failed to improve neuroprotection in association to corticosteroids in optic neuritis, likely for bias reasons (reviewed in 22). Of specific relation to the current case, it has been reported in NAION (27) (reviewed in 28) and traumatic ON injury (29 Rashad), and in one case of acute severe central RVO (CRVO) (Luscan and Roche, Société Francaise d’Ophtalmologie 2017 annual meeting). In addition EPO RPE gene therapy was recently suggested to prevent retinal degeneration induced by OS in a rodent model of dry Age Macular Degeneration (AMD) (30).

CASE REPORT PRESENTATION

This 54 years female patient was first visited on March 2019 4th, seeking for second opinion for ongoing vision deterioration OR on a daily basis, since around 3 weeks. Sub-central RVO (CRVO) OR had been diagnosed on February 27th; available SD-OCT macular volume was increased with epiretinal marked hyperreflectivity, one available Fluorescein angiography picture showed a non-filled superior CRVO, and a vast central ischemia involving the macular and paraoptic territories. Of note there was ON edema with a para-papillary hemorrage nasal to the disc on the available colour fundus picture.

At presentation on March 4, Best Corrected Visual Acuity (BCVA) was reduced at 20/100 OR (20/25 OS). The patient described periods of acutely excruciating retro-orbital pain in the OR. Intraocular pressure was normal, at 12 OR and 18 OS (pachymetry was at 490 microns in both eyes). The dilated fundus examination was similar to the previous color picture and did not disclose peripheral hemorrages recalling extended peripheral retinal ischemia. Humphrey Visual Field disclosed an altitudinal inferior scotoma and a peripheral inferior scotoma OR and was in the normal range OS, i.e. did not recall normal tension glaucoma OS (Fig. 1). There were no papillary drusen on the autofluorescence picture, ON volume was increased (11.77 mm3 OR versus 5.75 OS) on SD-OCT (Heidelberg Engineering®) OR, Retinal Nerve Fiber (RNFL) and RGC layers thicknesses were normal (Fig. 2). Marked epimacular hypereflectivity OR with foveolar depression inversion, moderately increased total volume and central foveolar thickness (CFT) (428 microns versus 328 OS), and a whitish aspect of the supero-temporal internal retinal layers recalling ischemic edema, were present (video 1). EDI CFT was incresead at 315 microns (versus 273 microns OS), with focal pachyvessels on the video mapping (video 1). OCT-Angiography disclosed focal perfusion defects in both the retinal and chorio-capillaris circulations (Fig. 3), and central alterations of the PR1 layer on en-face OCT(Fig. 4).

Altogether the clinical picture evoked a NAION with venous sub-occlusion, recalling Fraenkel’s et al early hypothesis of an ET interstitial diffusion-related venous vasoconstriction behind the lamina cribrosa (2), as much as a rupture of the BRB was present in the optic nerve area (hemorrage along the optic disc). Choroidal vascular drop-out was suggested by the severity and rapidity of the VF impairment (31). The extremely rapid development of a significant “epiretinal membrane”, that we interpreted as a reactive - and protective, in absence of cystoid macular edema (CME) - ET 2-induced astrocytic proliferation (reviewed in 32), was as an additional sign of severe ischemia.

The mention of the retro-orbital pain evoking a “ciliary angor”, the absence of any inflammatory syndrome and of the usual metabolic syndrome in the emergency blood test, oriented the etiology towards a FS. And indeed anamnesis collected many features of the FS, i.e. hypotension (“non dipper” profile with one symptomatic nocturnal episode of hypotension on the MAPA), migrains, hypersensitivity to cold, stress, noise, smells, and medicines, history of a spontaneously resolutive hydrops six months earlier, and of paroxystic episods of vertigo (which had driven a prior negative brain RMI investigation for Multiple Sclerosis, a frequent record among FS patients (33) and of paroxystic visual field alterations (7)(9), that were actually recorded several times along the follow-up.

The diagnosis of FS was eventually confirmed in the Ophthalmology Department in Basel University on April 10th, with elevated retinal venous pressure (20 to 25mmHg versus 10-15 OS) (4)(7)(9), reduced perfusion in the central retinal artery and veins on ocular Doppler (respectively 8.3 cm/second OR velocity versus 14.1 mmHg OS, and 3.1/second OR versus 5.9 cm OS), and impaired vasodilation upon flicker light-dependant shear stress on the Dynamic Vessel Analyser testing (7-9). In addition atherosclerotic plaques were absent on carotid Doppler.

On March 4th, the patient was at length informed about the FS, a possible off label rhEPO IVI, and a related written informed consent on the ratio risk-benefits was delivered.

By March 7th, she returned on an emergency basis because of vision worsening OR. VA was unchanged, intraocular pressure was at 13, but Visual Field showed a worsening of the central and inferior scotomas with a decreased foveolar threshold, from 33 to 29 decibels. SD-OCT showed a 10% increase in the CFT volume.

On the very same day, an off label rhEPO IVI OR (EPREX® 2000 units, 0,05 cc in a pre-filled syringe) was performed in the operating theater, i.e. the dose reported by Modarres et al (27), and twenty times inferior to the usual weekly intravenous dose for treatment of chronic anemia secondary to CKD. Intra venous acetazolamide (500 milligrams) was performed prior to the injection, to prevent any increase in intra-ocular pressure. The patient was discharged with a prescription of chlorydrate betaxolol (Betoptic® 0.5 %) two drops a day, and high dose daily magnesium supplementation (600 mgr).

Incidentally the patient developed bradycardia the day after, after altogether instillation of 4 drops of betaxolol only, that was replaced by acetazolamide drops, i.e. a typical hypersensitivity reaction to medications in the FS (7)(9).

Subjective vision improvement was recorded as early as D1 after injection. By March 18 th, eleven days post rhEPO IVI, BCVA was improved at 20/63, the altitudinal scotoma had resolved (Fig. 5), Posterior Vitreous Detachment had developed with a disturbing marked Weiss ring, optic disc swelling had decreased; vasculogenesis within the retinal plexi and some regression of PR1 alterations were visible on OCT-en face. Indeed by 11 days post EPO significant functional, neuronal and vascular rescue were observed, while the natural evolution had been seriously vision threatening.

However cystoid ME (CME) had developed (video 2). Indo Cyanin Green-Cine Video Angiography (ICG-CVA) OR, performed on March 23, i.e. 16 days after the rhEPO IVI, showed a persistent drop in ocular perfusion: ciliary and central retinal artery perfusion timings were dramatically delayed at respectively 21 and 25 seconds, central retinal vein perfusion initiated by 35 seconds, was pulsatile, and completed by 50 seconds only (video 3). Choroidal pachyveins matching the ones on SD-OCT video mapping were present in the temporal superior and inferior fields, and crossed the macula; capillary exclusion territories were present in the macula and around the optic disc.

By April 1, 23 days after the rhEPO injection, VA was unchanged, but CME and perfusion voids in the superficial deep capillary plexi and choriocapillaris were worsened, and optic disc swelling had recurred back to baseline, in a context of repeated episodes of systemic hypotension; and actually Nifepidin-Ratiopharm® oral drops (34), that had been delivered via a Temporary Use Authorization from the central Pharmacology Department in Assistance Publique Hopitaux de Paris, had had to be stopped because of hypersensitivity.

A second off label rhEPO IVI was performed in the same conditions on April 3, i.e. approximately one month after the first one.

Evolution was favourable as early as the day after EPO injection 2: VA was improved at 20/40, CME was reduced, and perfusion improved in the superficial retinal plexus as well as in the choriocapillaris. By week 4 after EPO injection 2, CME was much decreased, i.e. without anti VEGF injection. On august 19th, by week 18 after EPO 2, perfusion on ICG-CVA was greatly improved , with ciliary timing at 18 seconds, central retinal artery at 20 seconds and venous return from 23 to 36 seconds, still pulsatile. Capillary exclusion territories were visible in the macula and temporal to the macula after the capillary flood time that went on by 20.5 until 22.5 seconds (video 4); they were no longer persistent at intermediate and late timings.

Last complete follow-up was recorded on January 7, 2021, at 22 months from EPO injection 2. BCVA was at 20/40, ON volume had dropped at 7.46 mm3, a sequaelar superior deficit was present in the RNFL (Fig. 2) with some corresponding residual defects on the inferior para central Visual Field (Fig. 5), CFT was at 384 mm3 with an epimacular hyperreflectivity without ME, EDI CFT was dropped at 230 microns. Perfusion on ICG-CVA was not normalized, but even more improved, with ciliary timing at 15 seconds, central retinal artery at 16 seconds and venous return from 22 to 31 seconds, still pulsatile (video 5), indicating that VP was still above IOP. OCT-A showed persisting perfusion voids, especially at the optic disc and within the deep retinal capillary plexus. The latter were present at some degree in the OS as well (Fig. 6). Choriocapillaris and PR1 layer were dramatically improved.

Last recorded BCVA was at 20/32 by February 14, 2022, at 34 months from EPO 2. SD-OCT showed stable gliosis hypertrophy and mild alterations of the external layers (video 6).

Figure 1: Humphrey visual field at baseline on March 7th 2019, showing an altitudinal central scotoma, an inferior peripheral scotoma with a normal and symmetrical foveolar sensitivity threshold, and a normal visual field OS

Figure 2: Retinal Nerve Fiber (RNFL ) evolution from normal at baseline on March 2019 7th, to development of a superior sequellar deficit that remained stable on last follow-up.

Figure 3: OCT-Angiography at baseline on March 7th 2019, showing perfusion voids OR in the superior superficial retinal plexus and in the choriocapillaris.

Figure 4: OCT en face at baseline on March 7th 2019, showing PR1 layer deficits OR (artefacts in the superior half) compared to OS.

Figure 5: Humphrey visual field follow-ups : at follow-up 1 eleven days after rhEPO intra vitreal injection showing resolution of the altitudinal central scotoma and decrease of the inferior scotoma, and at last visual field follow-up on January 20th 2021, showing residual defects corresponding to the RNFL ones on Figure 2.

Figure 6: OCT Angiography performed on January 7th 2021, at 22 months from EPO injection 2, showing persisting perfusion voids, especially at the optic disc, and within the deep retinal capillary plexus, that were present at some degree in the OS as well.

Video 1 : SD-OCT video mapping OR at baseline on March 7th 2019, showing epiretinal hyperreflectivity and epiretinal membrane with foveolar depression inversion, ischemic edema in the internal and temporal to the disc superior retinal layers, and focal choroidal Haller pachyvessels with reduction in chorio-capillaris/Sattler layers.

Vedio: https://jmedcasereportsimages.org/articles/JCRMHS_1231_Vedio_1.mov

Video 2: SD-OCT video mapping OR at follow-up 1 eleven days after rhEPO intra vitreal injection on March 18th, showing epiretinal hyperreflectivity and epiretinal membrane with foveolar depression inversion, ischemic edema in the internal and temporal to the disc superior retinal layers, and development of central cystoid macular edema.

Vedio: https://jmedcasereportsimages.org/articles/JCRMHS_1231_Vedio_2.mov

Video 3 : Indo Cyanin Green-Cine Video Angiography OR, performed on March 23, i.e. 16 days after the rhEPO IVI, showing a persistent drop in ocular perfusion: ciliary and central retinal artery perfusion timings were dramatically delayed at respectively 21 and 25 seconds, central retinal vein perfusion initiated by 35 seconds, was pulsatile, and completed by 50 seconds only.

Vedio: https://jmedcasereportsimages.org/articles/JCRMHS_1231_Vedio_3.mov

Video 4 : Indo Cyanin Green-Cine Video Angiography OR, performed on August 19, i.e. by week 18 after EPO 2, showing greatly improved perfusion, with ciliary timing at 18 seconds, central retinal artery at 20 seconds and venous return from 23 to 36 seconds, still pulsatile. Capillary exclusion territories were visible in the macula and temporal to the macula after the capillary flood time that went on by 20.5 until 22.5 seconds.

Vedio: https://jmedcasereportsimages.org/articles/JCRMHS_1231_Vedio_4.mov

Video 5: Indo Cyanin Green-Cine Video Angiography OR, performed on January 7th, 2021, at 22 months from EPO injection 2: perfusion was not normalized, but even more improved, with ciliary timing at 15 seconds, central retinal artery at 16 seconds and venous return from 22 to 31 seconds, still pulsatile.

Vedio: https://jmedcasereportsimages.org/articles/JCRMHS_1231_Vedio_5.avi

Video 6 : SD-OCT video mapping at 34 months from EPO 2, showing stable gliosis hypertrophy and mild alterations of the external layers.

Vedio: https://jmedcasereportsimages.org/articles/JCRMHS_1231_Vedio_6.avi

DISCUSSION

What was striking in the initial clinical phenotype of CRVO was the contrast between the moderate venous dilation, and the intensity of ischemia, that were illustrating the pioneer hypothesis of Professor Flammer‘s team regarding the pivotal role of ET in VO (2), recently confirmed (3)(35), i.e. the local venous constriction backwards the lamina cribrosa, induced by diffusion of ET-1 within the vascular interstitium, in reaction to hypoxia. NAION was actually the primary and prevailing alteration, and ocular hypoperfusion was confirmed via ICG-CVA, as well as by the ocular Doppler performed in Basel. ICG-CVA confirmed the choroidal drop-out suggested by the severity of the VF impairment (31) and by OCT-A in the choriocapillaris. Venous pressure measurement, which instrumentation is now available (8), should become part of routine eye examination in case of RVO, as it is key to guide cases analysis and personalized therapeutical options.

Indeed, the endogenous EPO pathway is the dominant one activated by hypoxia and is synergetic with the VEGF pathway, and coherently it is expressed along to VEGF in the vitreous in human RVO (36). Diseases develop when the individual limiting stress threshold for efficient adaptative reactive capacity gets overwhelmed. In this case by Week 3 after symtoms onset, neuronal and vascular resilience mechanisms were no longer operative, but the BRB, compromised at the ON, was still maintained in the retina.

As mentioned in the introduction, the scientific rationale for the use of EPO was well demonstrated by that time, as well as the capacities of exogenous EPO to mimic endogenous EPO vasculogenesis, neurogenesis and synaptogenesis, restoration of the balance between ET-1 and NO. Improvement of chorioretinal blood flow was actually illustrated by the evolution of the choriocapillaris perfusion on repeated OCT-A and ICG-CVA. The anti-apoptotic effect of EPO (16) seems as much appropriate in case of RVO as the caspase-9 activation is possibly another overlooked co-factor (6).

All the conditions for translation into off label clinical use were present: severe vision loss with daily worsening and unlikely spontaneous favourable evolution, absence of toxicity in the human pilot studies, of contradictory comorbidities and co-medications, and of context of intraocular neovascularization that might be exacerbated by EPO (37).

Why didn’t we treat the onset of CME by March 18th, i.e. eleven days after EPO IVI 1, by anti-VEGF therapy, the “standard-of-care” in CME for RVO ?

In addition to the context of functional, neuronal and vascular improvements obviated by rhEPO IVI by that timing in the present case, actually anti VEGF therapy does not address the underlying causative pathology. Coherently, anti-VEGF IVI : 1) may not be efficient in improving vision in RVO, despite its efficiency in resolving/improving CME (usually requiring repeated injections), as shown in the Retain study (56% of eyes with resolved ME continued to loose vision)(quoted in (1) 2) eventually may be followed by serum ET-1 levels increase and VA reduction (in 25% of cases in a series of twenty eyes with BRVO) (38) and by increased areas of non perfusion in OCT-A (39). Rather did we perform a second hrEPO IVI, and actually we consider open the question whether the perfusion improvement, that was progressive, might have been accelerated/improved via repeated rhEPO IVI, on a three to four weeks basis.

The development of CME itself, involving a breakdown of the BRB, i.e. of part of the complex retinal armentorium resilience to hypoxia, was somewhat paradoxical in the context of improvement after the first EPO injection, as EPO restores the BRB (24), and as much as it was suggested that EPO inhibits glial osmotic swelling, one cause of ME, via VEGF induction (40). Possible explanations were: 1) the vascular hyperpermeability induced by the up-regulation of VEGF gene expression via EPO (41) 2) the ongoing causative disease, of chronic nature, that was obviated by the ICG-CVA and the Basel investigation, responsible for overwhelming the gliosis-dependant capacity of resilience to hypoxia 3) a combination of both. I/R seemed excluded: EPO precisely mimics hypoxic reconditioning as shown in over ten years publications, including in the retina (20), and as EPO therapy is part of the current strategy for stabilization of the endothelial glycocalix against I/R injury (42-43). An additional and not exclusive possible explanation was the potential antagonist action of EPO on GFAP astrocytes proliferation, as mentioned in the introduction (18), that might have counteracted the reactive protective hypertrophic gliosis, still fully operative prior to EPO injection, and that was eventually restored during the follow-up, where epiretinal hyperreflectivity without ME and ongoing chronic ischemia do coincide (Fig. 6 and video 6), as much as it is unlikely that EPO’s effect would exceed one month (cf infra). Inhibition of gliosis by EPO IVI might have been also part of the mechanism of rescue of RGG, compromised by gliosis in hypoxic conditions (44). Whatever the complex balance initially reached, then overwhelmed after EPO IVI 1, the challenge was rapidly overcome by the second EPO IVI without anti-VEGF injection, likely because the former was powerful enough to restore the threshold limit for resilience to hypoxia, that seemed no longer reached again during the relapse-free follow-up. Of note, this “epiretinal membrane “, which association to good vision is a proof of concept of its protective effect, must not be removed surgically, as it would suppress one of the mecanisms of resilience to hypoxia.

To our best knowledge, ICG-CVA was never reported in FS; it allows real time evaluation of the ocular perfusion and illustration of the universal rheological laws that control choroidal blood flow as well. Pachyveins recall a “reverse” veno-arteriolar reflex in the choroidal circulation, that is NO and autonomous nervous system-dependant, and that we suggested to be an adaptative choroidal microcirculation process to hypoxia (45). Their persistence during follow-up accounts for a persisting state of chronic ischemia.

The optimal timing for reperfusion via rhEPO in a non resolved issue:

in the case reported by Luscan and Roche, rhEPO IVI was performed on the very same day of disease onset, where it induced complete recovery from VA reduced at counting fingers at 1 meter, within 48 hours. This clinical human finding is on line with a recent rodent stroke study that established the timings for non lethal versus lethal ischemia of the neural and vascular lineages, and the optimized ones for beneficial reperfusion: the acute phase - from Day 1 where endothelial and neural cells are still preserved, to Day 7 where proliferation of pericytes and Progenitor Stem Cells are obtainable - and the chronic stage, up to Day 56, where vasculogenesis, neurogenesis and functional recovery are still possible, but with uncertain efficiency (46). In our particular case, PR rescue after rhEPO IVI 1 indicated that Week 3 was still timely. RhEPO IVI efficacy was shown to last between one (restoration of the BRB) and four weeks (antiapoptotic effect) in diabetic rats (24). The relapse after Week 3 post IVI 1 might indicate that it might be approximately the interval to be followed, should repeated injections be necessary.

The bilateral chronic perfusion defects on OCT-A at last follow-up indicate that both eyes remain in a condition of chronic ischemia and I/R, where endogenous EPO provides efficient ischemic pre-conditioning, but is potentially susceptible to be challenged during episodes of acute hypoxia that overwhelm the resilience threshold.

CONCLUSION

The present case advocates for individualized medicine with careful recording of the medical history, investigation of the systemic context, and exploiting of the available retinal multimodal imaging for accurate analytical interpretation of retinal diseases and their complex pathophysiology. The Flammer Syndrome is unfortunately overlooked in case of RVO; it should be suspected clinically in case of absence of the usual vascular and metabolic context, and in case of elevated RVP. RhEPO therapy is able to restore the beneficial endogenous EPO ischemic pre-conditioning in eyes submitted to challenging acute hypoxia episodes in addition to chronic ischemic stress, as in the Flammer Syndrome and fluctuating ocular blood flow, when it becomes compromised by the overwhelming of the hypoxic stress resilience threshold. The latter physiopathological explanation illuminates the cases of RVO where anti-VEGF therapy proved functionally inefficient, and/or worsened retinal ischemia. RhEPO therapy might be applied to other chronic ischemia and I/R conditions, as non neo-vascular Age Macular Degeneration (AMD), and actually EPO was listed in 2020 among the nineteen promising molecules in AMD in a pooling of four thousands (47).

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thientuepharmajsc · 1 year

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Silymarin – Chiết xuất Cây Kế Sữa | Cao khô Kế Sữa (Milk Thistle extract) là hoạt chất chính được chiết xuất từ cây kế sữa, hay còn được gọi là cây cúc gai đã được chứng minh lâm sàn là hoạt chất có tác dụng cho gan tôm tốt nhất trong số tất cả những hoạt chất có chức năng tương tự.

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Silymarin nguyên liệu có dạng bột có màu nâu và được sử dụng như là một chất bổ sung vào trong rất nhiều các loại thực phẩm, đồ ăn và thức uống khác nhau. Các nhà nghiên cứu khuyến cáo nên sử dụng hợp chất này với liều lượng khoảng 80mg/ngày.

2. Phân biệt Silymarin thường và Silymarin Phytosome®

Silymarin là một Flavonoid (chất chuyển hóa trung gian của thực vật), là một nhóm các thành phần được chiết xuất từ cây kế sữa (cây cúc gai). Trong số các thành phần đó, Silybin là hoạt chất chính, đã được chứng minh lâm sàn là có tác dụng vượt trội dành cho gan, và là hoạt chất có tác dụng cho gan tốt nhất trong số tất cả những hoạt chất có chức năng tương tự. Hiện nay, Silymarin ngoài thị trường sẽ có hai loại chính:

Silymarin thông thường là Silymarin có chứa Silybin được chiết xuất bằng phương pháp thông thường, đây là các Silybin tự do. Ưu điểm của loại Silymarin thông thường này là chúng có giá thành tương đối rẻ, thường được sử dụng để làm nguyên liệu cho sản xuất Thuốc Thủy Sản, Thuốc Thú Y hoặc dùng để phối trộn với Thức Ăn Chăn Nuôi (Thức ăn thủy sản, Thức ăn chăn nuôi gia súc, gia cầm).

Silymarin Phytosome® là Silymarin có chứa Silybin được chiết xuất bằng công nghệ Phytosome, đây còn được gọi là Silybin Phytosome. Công nghệ Phytosomes® là công nghệ dùng để chiết xuất các loại thảo dược, làm cao dược liệu tiên tiến nhất hiện nay.

Công nghệ Phytosome giúp các phân tử Silybin liên kết chặt chẽ với màng photpholipid của đậu nành và giúp cải thiện hoạt tính của Silybin lên mức tối ưu nhất. Chính vì thế, Silymarin Phytosome (hay còn được gọi là Silybin Phytosome) có giá thành tương đối cao, được sử dụng chủ yếu để làm nguyên liệu sản xuất dược như là nguyên liệu Thuốc và Thực phẩm chức năng.

3. Tác dụng của Silymarin trong việc bảo vệ và phục hồi chức năng gan

Silymarin đã được chứng minh hiệu quả của nó qua nhiều nghiên cứu lâm sàng khác nhau. Ngoài ra, Indena cũng đã tiến hành nhiều nghiên cứu để kiểm tra dược tính và độc tính của nó để đảm bảo sự hiệu quả và an toàn khi sử dụng.

Liều dùng của Silybin Tế bàoTác dụng của SilybinTừ 5 đến 50 µmol/LTế bào ganỨc chế tín hiệu do trung gian NF-κBTừ 10 đến 100 μmol/LTế bào ganGiảm sự hình thành các chất chứa từ ty thể, tạo phức chelat với kim loạiHepG2Giảm sự hình thành các anion oxy hóaTrung bình: 15 µmol/LTế bào biểu môỨc chế phosphoryl hóa IκBαTiểu cầuỨc chế protein kinaseTế bào ung thưỨc chế c-jun N-terminal kinaseĐại thực bàoỨc chế hình thành leukotrieneTế bào hình saoỨc chế giải phóng cytochrome cHepG2Ức chế phosphoryl hóa ERK, MEK, Raf; ức chế giải phóng caspase 9 và 3, IL-8; ức chế tín hiệu do trung gian PDGF và TGF-beta; giảm MMP2; tăng TIMP2; ức chế sự nhân lên của HCVLiều trung bình có tác dụng được ghi lại: 20 μmol/LTế bào KupfferGiảm sự sản xuất NOTế bào bạch cầu monoDọn các gốc tự do lipodienyl, methyl, trichloromethylTế bào biểu môGiảm nồng độ các hydrogen peroxideTế bào ung thưỨc chế quá trình peroxide hóa lipid màng tế bào

4. Ứng dụng của Silymarin trong việc phục hồi và bảo vệ gan tôm

Hiện nay, chưa có một nghiên cứu hay báo cáo cụ thể nào (kể cả trong nước và ngoài nước) trong việc ứng dụng Silymarin để phục hồi và bảo vệ gan tôm. Tuy nhiên, dựa trên các nghiên cứu về tác dụng phục hồi và bảo vệ gan của Silybin (hoạt chất chính trong Silymarin) lên gan chuột đã bị tổn thương, chúng ta có thể suy ra, Silymarin cũng có tác dụng tương tự đối với gan tôm nói riêng và gan của các loài thủy sản nói chung.

Silymarin đã được nghiên cứu lâm sàng và ứng dụng để phục hồi và bảo vệ gan cho người. Chính vì thế, hoạt chất này rất an toàn khi sử dụng cho thủy sản, đặc biệt là trong việc phục hồi và bảo vệ gan tôm. Một số công dụng chính khi dùng Silymarin cho gan tôm như sau:

Tăng cường hệ miễn dịch cho tôm, tăng cường chức năng gan tôm.

Phòng bệnh và điều trị các bệnh về gan trên tôm như sưng gan, teo gan, vàng gan, hoại tử gan tụy, nhiễm độc tố gan, …

Chữa bệnh sưng gan, phù nề gan thận, nhiễm độc tố gan.

Chống hoại tử gan tụy, giúp lọc máu, chống co thắt đường ruột tôm.

Chống rụng râu, mòn đuôi, đề kháng mạnh khi môi trường có sự biến động đột ngột.

Giảm tình trạng tôm chết đột ngột không rõ nguyên nhân.

Bên cạnh đó, hiện tại chưa có nghiên cứu khoa học nào cho thấy liều lượng cụ thể khi dùng silymarin để trộn cho tôm ăn. Nhưng theo thử nghiệm thực tế của Công Ty Thiên Tuế, liều lượng silymarin khi trộn cho tôm ăn có thể tham khảo như sau:

Tôm dưới 15 ngày: 3 gam cho 1 kg thức ăn.

Tôm từ 15 ngày đến 30 ngày: 6 gam cho 1 kg thức ăn.

Tôm từ 30 ngày đến 45 ngày: 10 gam cho 1 kg thức ăn.

Tôm trên 45 ngày: 15 gam cho 1 kg thức ăn.

Silymarin là một hoạt chất rất mạnh dành cho việc phục hồi và để bảo vệ gan tôm, chính vì thế, silymarin cho gan tôm thường được khuyến cáo là nên dùng định kì, xuyên suốt từ lúc mới thả đến hết vụ. Trong trường hợp tôm bị các bệnh gan, khả năng ăn thức ăn kém, người nuôi có thể hòa tan silymarin với nước rồi tạt thẳng xuống ao để đảm bảo tôm có thể hấp thụ silymarin một cách tốt nhất.

Chính vì những lợi ích cực kì đặc biệt dành cho gan như vậy mà silymarin dần trở thành một trong những hoạt chất cực kì quan trọng dùng trong nuôi tôm nói riêng và trong nuôi trồng thủy sản nói chung. Silymarin hiện tại đang được thương mại ở hai dạng chính:

Một là silymarin nguyên liệu, có nguồn gốc xuất sứ ở Việt Nam, Trung Quốc hoặc các nước Châu Âu như Đức, Ý.

Hai là thuốc thủy sản có chứa Silymarin. Đây là dạng silymarin đã được phối trộn, kết hợp với các thành phần khác để tạo thành dạng thành phẩm, ngoài chức năng phục hồi và bảo vệ gan tôm, các loại thuốc thủy sản có chứa silymarin như thế này thường sẽ đi kèm thêm nhiều công dụng khác dành cho tôm

--------- Follow page Thientue Pharma JSC - Bộ phận THỦY SẢN để đón đọc thêm nhiều thông tin hữu ích về ngành nuôi tôm. Follow page Thientue Pharma JSC - Nguyên liệu dược liệu để đón đọc thêm nhiều thông tin hữu ích về sức khỏe con người. #ThientuePharmaJSC #duocphamthientue #congtythientue#aquaculture #thuysan #livestock #channuoi #tpcn #functionalfoods#silymarin #chietxuatkesua #caykesua #caycucgai #sylibin #phytosome --------- Thientue Pharma JSC - Hợp Tác Chân Thành Công Ty Cổ Phần Dược Phẩm Thiên Tuế (Thientue Pharma JSC) chuyên sản xuất và phân phối các loại Cao dược liệu, Chiết xuất thảo dược, Chế phẩm sinh học (men vi sinh, nguyên liệu sinh học) và các hoạt chất nhập khẩu chuyên dụng dành cho thủy sản. VPĐD: 56/30 Tân Thới Nhất 17, KP4, P. TNT, Q. 12, Tp. HCM 71510S ĐT: 028.6267.707

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latestsmarkettrends · 1 year

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moleculardepot · 1 year

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Granzyme B Inhibitor II

Granzyme B Inhibitor II Catalog number: B2013840 Lot number: Batch Dependent Expiration Date: Batch dependent Amount: 1 mg Molecular Weight or Concentration: 502.52 Supplied as: Lyophilized Solid Applications: molecular tool for various biochemical applications Storage: −20°C Keywords: Granzyme B Inhibitor II, Caspase-8 Inhibitor I, Ac-IETD-CHO Grade: Biotechnology grade. All products are highly…

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soumyafwr · 9 months

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https://twikkers.nl/blogs/174365/Caspase-8-Market-Overview-Competitive-Analysis-and-Forecast-2031

Caspase 8 Market Overview, Competitive Analysis and Forecast 2031

#Caspase 8 Market #Caspase 8 Market Scope #Caspase 8 Market Report #Caspase 8 Market Research

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moleculardepot · 2 years

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Caspase 8 Processing Enzyme Substrate

Caspase 8 Processing Enzyme Substrate Catalog number: B2013010 Lot number: Batch Dependent Expiration Date: Batch dependent Amount: 2 mg Molecular Weight or Concentration: 730.7 g/mol Supplied as: Lyophilized Powder Applications: molecular tool for various biochemical applications Storage: -20°C Keywords: Granzyme B Substrate, Caspase 3 Processing Enzyme Substrate, Caspase 8 Processing Enzyme…

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wwwsarms4musclecom · 2 years

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cas: 1890208-58-8 Compound 7P Strongest Nootropic

IS COMPOUND 7P A BRAIN REGENERATIVE COMPOUND?

The neurons in the adult central nervous system (CNS) fail to regenerate axons after injury, which accounts in part for the poor functional recovery in patients with traumatic and neurodegenerative diseases (1).

The failure of axon regeneration is attributable to a growth-inhibiting environment involving myelin-associated inhibitors (Nogo-A, myelin-associated glycoprotein, and oligodendrocyte/myelin glycoprotein) and glial scar at the injury site.However, neutralizing and/or removing such inhibitory factors are insufficient to promote long-distance axon regeneration (1).‍

In this study, in an effort to discover new pharmacological modalities to aid in axon regeneration, we employed phenotypic cell-based screens that allow visual assessment and quantitative measurement of neurite outgrowth in vitro. The phenotypic screening campaign and chemical modification efforts led to identification of compound 7p that enhances neurite outgrowth in cultured primary neurons derived from the hippocampus, cerebral cortex, and retina and that induces optic nerve regeneration in an animal model of optic nerve injury. Although it needs to be determined how the compound stimulates axon growth in vivo, our results should provide further insight into the treatment strategies for clinical conditions associated with a loss of axon.

Another report using phenotypic cell-based screen of chemical libraries and structure-activity-guided optimization resulted in the identification of compound 7p which promotes neurite outgrowth of cultured primary neurons derived from the hippocampus, cerebral cortex, and retina. in an animal model of optic nerve injury, compound 7p was shown to induce growth of gap-43 positive axons, indicating that the in vitro neurite outgrowth activity of compound 7p translates into stimulation of axon regeneration in vivo.further optimization of compound 7p and elucidation of the mechanisms by which it elicits axon regeneration in vivo will provide a rational basis for fut

Compound 7p (2-[(2-methoxyphenyl)[(4-methyl phenyl) sulfonyl] amino]-N-(4-methoxy-3-pyridinyl) acetamide) showed the highest activity against cervical cancer cells. In a nude mouse xenograft model inoculated with HeLa cells, 7p showed dose-dependent inhibition of cervical tumour growth. Histopathological examination of excised tumour-bearing tissues showed that 7p improved the microstructure in a dose-dependent manner. Compound 7p also increased the proportions of HeLa cells in G0/G1 and S-phase and significantly decreased that of G2/M-phase. The effects of 7p on C-caspase-3, C-caspase-9, Bcl-2 and Bax expression in HeLa cells were also determined.

Compound 7p was developed as one in series of compounds with the aim of identifying dual-acting thromboxane receptor antagonist/synthase inhibitors. In fact compound 7p shows selectivity for prostaglandin I2 synthase (PTGIS, CYP8A1) over thromboxane synthase (CYP5A1).

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petnews2day · 2 years

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Increased autophagy leads to decreased apoptosis during β-thalassaemic mouse and patient erythropoiesis

New Post has been published on https://petnews2day.com/pet-news/small-pet-news/increased-autophagy-leads-to-decreased-apoptosis-during-%ce%b2-thalassaemic-mouse-and-patient-erythropoiesis/

Increased autophagy leads to decreased apoptosis during β-thalassaemic mouse and patient erythropoiesis

Animals

This study was approved by the Mahidol University Animal Care and Use Committee (approval number MU-ACUC 2009/001). All methods were performed in accordance with the relevant guidelines and regulations. Wild type mice and βIVS2-654-thalassaemia in C57Bl/6 background mice20 were employed in the present study. Murine bone marrow cells were harvested from femurs and tibias as described in a previous study21. All methods are reported in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020).

Flow cytometric analysis of murine erythroid apoptosis

Erythroid subpopulations were defined as described in previous studies18,26. Whole bone marrow samples were stained with fluorochrome conjugated monoclonal antibodies specific to CD71 (transferrin receptor, BD Biosciences, San Jose, CA) and TER119 (red cell marker, BD Biosciences). Then, samples were combined with a third-color fluorescent channel staining such as fluorochrome conjugated annexin V (phosphatidylserine marker, BD Biosciences) or 100 nM 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) (Sigma-Aldrich, St. Louis, MO) to determine mitochondrial transmembrane potential. For determination of activated caspase 8 and caspase 9 in living cells, bone marrow samples (2 × 106 cells) were incubated with either fluorescein isothiocyanate (FITC)-conjugated IETD-FMK (for caspase 8, Calbiochem, San Diego, CA) or sulforhodamine (Red)-conjugated LEHD-FMK (for caspase 9, Calbiochem) following the manufacturer’s recommendation. The cocktail of monoclonal antibodies that was used in this study are shown in Supplementary Table 1. Data of 100,000 events was acquired and analysed using a BD FACSCalibur flow cytometer and CellQuest Pro™ software (BD Biosciences). K562 erythroleukemic cells treated with 193.8 mM hydrogen peroxide (H2O2) for 15 min at 37 °C, 5% CO2 were used as a control.

Murine bone marrow erythroblast isolation

CD45− erythroblasts and CD45–CD71+ erythroblasts from bone marrow samples were isolated using a MACS cell separation system (Miltenyi Biotec, Bergen Gladbach, Germany) as the manufacturer’s instructions. The purity of murine CD45–CD71+ bone marrow erythroblasts was 73–91% as measured by flow cytometry.

Transmission electron microscopic analysis of murine erythroid autophagy

Bone marrow erythroblasts were harvested and analysed for morphology and LC3 expression using a transmission electron microscope41. CD45– bone marrow erythroid cells (1 × 107 cells) were fixed in 2.5% glutaraldehyde solution for 4 h at 4 °C, then, washed with Sorenson’s phosphate buffer and stained with 1% Caulfield’s osmium tetroxide supplemented with sucrose for 1 h. Samples were dehydrated with different concentrations of ethanol and finally propylene oxide, then, embedded in Embed 812 resin BEEM capsules (Electron Microscopy Science, Hatfield, PA) at 65–80 °C for overnight. Thin section (60–90 nm) was collected and mounted on copper grids, stained with uranyl acetate, and counterstained with lead citrate. Grids were observed and images were obtained using a transmission electron microscope (Hitachi H-7100, Hitachi High-Tech Science Corporation, Tokyo, Japan) operated at 100 kV. Images were captured at 4000 × magnification for 10 fields per sample by sequential field collection. Autophagic vacuoles/autolysosomes was defined as membrane-bound vacuole containing electron-dense cytoplasmic material and/or organelles at various stages of degradation. The electron-lucent cleft between the two limiting membranes was used to aid identification. The area of autophagic vacuoles and the total cytoplasmic area of erythroblasts were measured using the Scion Image Beta 4.02 analysis software (Scion Corporation, Chicago, IL). A total of 50 cells per sample were analysed.

For LC3 expression determination by immunogold staining41, a thin section of CD45– bone marrow erythroid cells from β-thalassaemic mice processed for transmission electron microscopy were collected and mounted on nickel grids. Grids were blocked with 0.5% BSA-C (Aurion, Wageningen, Netherlands), and then stained with a rabbit anti-LC3 polyclonal antibody (Aurion) as a primary antibody and gold-conjugated F(ab)2 fragments of goat anti-rabbit IgG (Aurion) as a secondary antibody, and counterstained with uranyl acetate. Control samples used rabbit serum (Dako, Glostrup, Denmark) in place of the primary antibody were used as a negative control. Images were captured using a transmission electron microscope (Hitachi H-7100) operated at 100 kV.

Confocal microscopic analysis of murine erythroid autophagy

CD45– bone marrow erythroid cells (3 × 105 cells) were fixed with 4% paraformaldehyde and blocked with 1% BSA-C solution (Aurion) for 1 h. Subsequently, samples were permeabilized with 0.3% Triton X-100 in phosphate buffered saline and incubated with a rabbit anti-LC3 polyclonal antibody (Novus Biologicals, Littleton, CO), then, subsequently a Cy5-conjugated goat anti-rabbit IgG (H+L) F(ab’)2 fragments (Invitrogen, Carlsbad, CA). After incubation and wash, samples were stained with FITC conjugated rat anti-LAMP-1 monoclonal antibody (Biolegend, San Diego, CA). Fluorescent signal was observed using an Olympus FluoView 1000 confocal microscope (Olympus, Tokyo, Japan). Illustration was captured using a 60 × objective lens for 10 field per sample by sequential field collection and discard 10 fields. A total of 50 cells per sample were analysed. Image analysis and calculation of Pearson’s correlation coefficients and confidence intervals were undertaken as previously described17,42. Autophagosomes with punctate LC3 expression in erythroblasts were count and calculated as the percentage of autophagic cells in total erythroid cells (Supplementary Fig. S4).

Inhibition autophagic flux of murine erythroblasts

CD45–CD71+ bone marrow erythroblasts were cultured in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 20% fetal bovine serum (FBS) with or without 100 μM chloroquine at 37 °C, 5% CO2. Chloroquine was dissolved in culture medium and filtered through a 0.2 μM filter before use. The cells were collected at 3 h. after treatment for analysis of LC3-I and LC3-II by western blot43 and at 24 h. for determination of PS expose by flow cytometry.

Western blot analysis of erythroid autophagy

CD45–CD71+ bone marrow erythroid cells were lysed and sonicated in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% SDS and 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma-Aldrich). A total of 50 μg protein of the lysates was separated through 15% denaturing polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories). Primary antibodies used were a rabbit anti-caspase 3 polyclonal antibody (Cell Signaling Technology, Danvers, MA), a rabbit anti-LC3 polyclonal antibody (Abcam, Cambridge, UK) and a rabbit anti-β-actin monoclonal antibody (Sigma-Aldrich). Secondary antibody was used was a horseradish peroxidase conjugated goat anti-rabbit IgG (Sigma-Aldrich). The densitometric analysis was performed using ImageJ software (The National Institutes of Health, MD).

Human bone marrow collection

This study was performed in accordance with the Helsinki declaration and was approved by the Mahidol University Institutional Review Board (approval number MU-CIRB 2014/031.1703). Written informed consent was obtained from all individual participants in this study. Patients under treatment with hydroxyurea, aspirin, antibiotics, anti-depressants, beta-blockers and anti-platelets were excluded, and no participant had been hospitalised or transfused within 4 weeks of sample collection. Human bone marrow samples were collected from 6 β-thalassaemia/HbE patients and 3 control subjects into citrate phosphate dextrose adenine solution anticoagulant by using the bone marrow aspiration technique. Samples were processed within 1 h after aspiration. All methods were performed in accordance with the relevant guidelines and regulations. Haematological parameters are shown in Supplementary Table 2.

Human bone marrow erythroblast isolation

CD45– erythroblasts from bone marrow samples were isolated using a MACS cell separation system (Miltenyi Biotec) as the manufacturer’s instructions. The purity of human CD45– bone marrow erythroblasts was 45–70% as measured by flow cytometry.

Flow cytometric analysis of human erythroid apoptosis

Erythroid subpopulations were defined as described in previous studies17,27. Whole bone marrow samples were stained with fluorochrome conjugated monoclonal antibodies specific to CD45 (leukocyte marker, BD Biosciences), CD71 (BD Biosciences) and glycophorin A (GPA, red cell marker, BD Biosciences). Then, samples were combined with a fourth-color fluorescent channel staining such as fluorochrome conjugated annexin V (BD Biosciences) or 50 nM tetramethylrhodamine ethyl ester perchlorate (TMRE) (Sigma-Aldrich) for mitochondrial transmembrane potential. Whole bone marrow samples were incubated with 100 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 30 min at 37 °C, 5% CO2 for direct disruption of mitochondrial transmembrane potential and this was used as a control.

For intracellular staining to determine active caspase 3, whole bone marrow samples were incubated with cold-cytoFix/Perm (BD Biosciences) for 10 min at 4 °C and then were stained with fluorochrome conjugated monoclonal antibodies specific to CD45, CD71, GPA and either activated caspase 3 (BD Biosciences) or isotype control for 30 min at 4 °C. Data of 100,000 events was acquired and analysed using a BD FACSCalibur flow cytometer and CellQuest Pro™ software. Bone marrow sample treated with 193.8 mM H2O2 for 15 min at 37 °C, 5% CO2 was used as positive control. The cocktail of monoclonal antibodies that was used in this study are shown in Supplementary Table 1.

Human erythroid cell autophagic analysis

CD45– human bone marrow erythroblasts were isolated and analysed co-localization of LC3/LAMP-1 using confocal microscope17,42. CD45– human bone marrow erythroblasts (3 × 105t5 cells) were fixed and stained with a rabbit anti-LC3 polyclonal antibody (Novus Biologicals), then, subsequently a Cy5-conjugated goat anti-rabbit IgG (H+L) F(ab’)2 fragments (Invitrogen) as secondary antibody, after incubation and wash, samples were incubated with a FITC conjugated rat anti-LAMP-1 monoclonal antibody (Biolegend). After incubation and wash, samples were stained with 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI, Molecular Probes, Eugene, OR). Images were captured using a 60 × objective lens for ≥ 10 field per sample. A total of 50 erythroid cells per sample were analysed. Fluorescent signal was observed and analysed as described in the section of “Confocal microscopic analysis of murine erythroid autophagy” (Supplementary Figs. S8 and S9).

Statistical analysis

Data were analyzed using SPSS Version 18.0 (SPSS Inc., Chicago, IL). Comparisons between parameters which analysed using flow cytometry and cell counting were evaluated by Mann–Whitney U test. Comparisons between parameters which image analysis including autophagic vacuole per cytoplasmic area ratio and densitometric analysis of protein expression using Western blot were evaluated by independent Student’s t-tests. The threshold for statistical significance for all comparisons was P < 0.05.

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lupinepublishers · 3 years

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lupine publishers|Circ_0001529 Expression Abnormalities Mediate the miR-578 CDC25A Axis Involved in the Growth of Ovarian Cancer

Circ_0001529 Expression Abnormalities Mediate the miR-578 CDC25A Axis Involved in the Growth of Ovarian Cancer

Abstract Purpose: To investigate the effect of Circ_0001529-mediated miR-578-CDC25A axis on the growth of ovarian cancer cells in vitro and in vivo. Method: The expression differences of Circ_0001529 in ovarian cancer tissues and paraneoplastic tissues were first analyzed; Circ_0001529 overexpression plasmids and interference plasmids were constructed and transfected into SKOV3 cells and SiHa cells, respectively, followed by MTT assay, flow cytometry, and immunofluorescence to detect cell growth, proliferation, and apoptosis. The binding relationship between Circ_0001529 and miR-578, or miR-578 and CDC25A was verified by online prediction site and dual luciferase assay, and CDC25A overexpression plasmid and interference plasmid were constructed and transfected into SKOV3 cells and SiHa cells, respectively. qRT-PCR and Western Blot were used to to detect the expression of ACP5, followed by cell growth as well as apoptosis. A mouse xenograft tumor model was constructed, and the growth of ovarian cancer cells in vivo was assessed by calculating tumor volume growth with KI67 and Caspase-3 immunohistochemistry. Result: Circ_0001529 was highly expressed in ovarian cancer tissues and cells; silencing Circ_0001529 inhibited ovarian cancer cell growth and promoted apoptosis in vitro and in vivo. circ_0001529 could bind to miR-578 target, and miR-578 bound to CDC25A mRNA target. Overexpression of Circ_0001529 promotes the expression of CDC25A and promotes the growth and inhibits the apop- tosis of ovarian cancer cells. CDC25A can upregulate the ERK/MAPK signaling pathway, thus promoting ovarian carcinogenesis. Conclusion: circRNA_0001529 promotes CDC25A expression through targeted binding of miR-578, thereby promoting the growth and metastasis of ovarian cancer cells. Key words: ACP5; Circ_0001529; Lung Carcinoma; Kalpan-Meier Analysis; Proliferation; Apoptosis; Erk/Mapk Signaling Pathway

Introduction Ovarian cancer (OC) is one of the deadliest gynecological cancers, with a significant fatality rate. It has a poor prognosis due to late diagnosis and medication resistance [1]. In the treatment of ovarian cancer, it is critical to distinguish between patients who will benefit from primary full cytoreductive surgery and those who will benefit more from neoadjuvant chemotherapy [2]. TK1 could be used as a biomarker to diagnose ovarian cancer. When it comes to differentiating malignant ovarian tumors from benign ones, ROMI outperforms ROMA [3]. The use of cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) has been demonstrated to be effective in a variety of cancers, and it may also provide a prognosis benefit for patients with advanced ovarian cancer [4]. The intracellular signal transducer and regulator of gene expression and protein turnover, Focal adhesion Kinase (FAK), increases tumor progression and is a promising therapeutic target for a variety of cancers, including ovarian cancer. [5] There have been some results suggesting that ERRα may promote ovarian cancer progression and may serve as a promising predictive biomarker [6]. Ovarian Cancer (OC) is a type of gynecological cancer with high mortality rate, and Circ_000152. (PM9 plays an important role in it. However, the clinical significance of circular RNA and its molecular mechanism in OC are mostly unknown. Circ_0001529 (hsa_circAFF4_016), is a circular RNA formed by reverse shearing of AFF4, is a circular RNA newly discovered by us [7] Circular RNAs (circRNAs) are increasingly recognized as important regulators in cancer including ovarian cancer (OC). This work focuses on the effects of circ_0000745 on the OC development of and molecules involved [8]. Imaging intracellular microRNAs (miRNAs) demonstrated an essential role in exposing their biological and pathological functions [9]. The induction of microribonucleic acid (miRNA) in cells is of great significance for early disease diagnosis and treatment monitoring. DNA is an interesting building material that can be programmed into components with rigid and branched structures and is particularly suitable for intracellular biomolecular imaging or therapeutic drug delivery [10]. Moreover, it has been shown that targeting the hsa_circ_0005785/miR-578/ APRIL regulatory pathway may be a promising diagnostic and therapeutic strategy in the clinical practice of HCC [11]. Similarly, for the ovarian cancer we studied, we combined Circ_0001529 with miR-578 targeting to see if it could promote cancer cell changes and metastasis functionally. Given the importance of silencing Circ_0001529 in ovarian cancer treatment, we aimed to identify an excellent inhibitor and investigate its potential therapeutic effects, thereby knocking down the role of Circ_0001529 expression to promote ovarian cancer cell growth and inhibit its apoptosis. Circular RNA (circRNA) is recently found to participate in the regulation of tumor progression, including ovarian cancer. Methods Tissue samples Eighty patients with ovarian cancer diagnosed and treated in our hospital from September 2012 to June 2013 were selected from surgically resected cancerous tissue and normal paracancerous tissue (at least 2 cm away from the ovarian cancerous tissue), with a median age of 49-79 years and a mean age of (64.9 ± 7.3) years. And all patients were followed up every three months for five years. Inclusion criteria: 1, all patients were diagnosed with ovarian cancer by postoperative pathological examination; 2, all patients did not receive radiotherapy or chemotherapy before surgery; 3, clinical data were complete. Exclusion criteria: 1, combined with chronic systemic diseases; 2, combined with other malignant tumors. The study was approved by our ethics committee and supervised by our ethics committee, and the subjects all signed the informed consent form while enjoying the right to information. Cell Culture Human ovarian cancer cell lines Hela, SKOV3, SiHa, SPC-A-1, L9981 and human normal ovarian epithelial cells

HOSE-1 were purchased from the Tumor Cell Bank of China Medical College (Beijing, China), and subsequently inoculated into cell culture dishes at a density of 1×10 5 cells/cm2 using RPMI-1640 medium containing 10% fetal bovine serum, CM7-1 medium, F12K medium and LHC-9 medium (all purchased from Gibco, Grand Island, NY,USA), and cultured at 37°C , 5% CO2 for 48 h until cell growth was fused to 80%-90%, digested with 0.025% trypsin (Gibco), and passaged. qRT-PCR Total RNA of tumor tissues and cells was used using RNAiso Plus (TAKARA, Otsu, Shiga, Japan) and Trizol LS Reagent (TAKARA, Otsu, Shiga, Japan), respectively. The reliability of the obtained RNA was subsequently verified using formaldehyde denaturation electrophoresis assay to continue the subsequent experiments. Reverse transcription polymerase chain reaction (RT-PCR) experiments were then performed using the PrimeScript TMRT kit (TAKARA, Otsu, Shiga, Japan) strictly according to the instructions. The mRNA expression levels were quantified by standard real-time quantitative PCR methods using SYBR Premix Ex Taq (TAKARA, Otsu, Shiga, Japan). gAPDH was used as a reference gene. The primers for each group are shown in Table 1.

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