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AAV expressing an mTOR-inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity.

ABSTRACT: Expanding on previous demonstrations of the therapeutic effects of adeno-associated virus (AAV) carrying small-hairpin RNA (shRNA) in downregulating the mechanistic target of rapamycin (mTOR) in in vivo retinal vascular disorders, vascular endothelial growth factor (VEGF)-stimulated endothelial cells were treated with AAV2-shmTOR to examine the role of mTOR inhibition in retinal angiogenesis. AAV2-shmTOR exposure significantly reduced mTOR expression in human umbilical vein endothelial cells (HUVECs) and decreased downstream signaling cascades of mTOR complex 1 (mTORC1) and mTORC2 under VEGF treatment. Moreover, the angiogenic potential of VEGF was significantly inhibited by AAV2-shmTOR, which preserved endothelial integrity by maintaining tight junctions between HUVECs. These data thus support previous in vivo studies and provide evidence that AAV2-shmTOR induces therapeutic effects by inhibiting the neovascularization of endothelial cells.

SUBMITTER: Cha S

PROVIDER: S-EPMC8727948 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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AAV expressing an mTOR-inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity.

Cha Seho S Seo Won-Il WI Woo Ha-Na HN Kim Hee Jong HJ Lee Steven Hyun Seung SHS Kim Jin J Choi Jun-Sub JS Park Keerang K Lee Joo Yong JY Lee Beom Jun BJ Lee Heuiran H

FEBS open bio 20211021 1

Expanding on previous demonstrations of the therapeutic effects of adeno-associated virus (AAV) carrying small-hairpin RNA (shRNA) in downregulating the mechanistic target of rapamycin (mTOR) in in vivo retinal vascular disorders, vascular endothelial growth factor (VEGF)-stimulated endothelial cells were treated with AAV2-shmTOR to examine the role of mTOR inhibition in retinal angiogenesis. AAV2-shmTOR exposure significantly reduced mTOR expression in human umbilical vein endothelial cells (HU ...[more]

PMID: 34431239

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Project description:Glaucoma is the leading cause of irreversible blindness and is characterized by slow and progressive degeneration of the optic nerve head axons and retinal ganglion cell (RGC), leading to loss of visual function. Although oxidative stress and/or alteration of mitochondrial (mt) dynamics induced by elevated intraocular pressure (IOP) are associated with this neurodegenerative disease, the mechanisms that regulate mt dysfunction-mediated glaucomatous neurodegeneration are poorly understood. Using a mouse model of glaucoma, DBA/2J (D2), which spontaneously develops elevated IOP, as well as an in vitro RGC culture system, we show here that oxidative stress, as evidenced by increasing superoxide dismutase 2 (SOD2) and mt transcription factor A (Tfam) protein expression, triggers mt fission and loss by increasing dynamin-related protein 1 (DRP1) in the retina of glaucomatous D2 mice as well as in cultured RGCs exposed to elevated hydrostatic pressure in vitro. DRP1 inhibition by overexpressing DRP1 K38A mutant blocks mt fission and triggers a subsequent reduction of oxidative stress, as evidenced by decreasing SOD2 and Tfam protein expression. DRP1 inhibition promotes RGC survival by increasing phosphorylation of Bad at serine 112 in the retina and preserves RGC axons by maintaining mt integrity in the glial lamina of glaucomatous D2 mice. These findings demonstrate an important vicious cycle involved in glaucomatous neurodegeneration that starts with elevated IOP producing oxidative stress; the oxidative stress then leads to mt fission and a specific form of mt dysfunction that generates further oxidative stress, thus perpetuating the cycle. Our findings suggest that DRP1 is a potential therapeutic target for ameliorating oxidative stress-mediated mt fission and dysfunction in RGC and its axons during glaucomatous neurodegeneration. Thus, DRP1 inhibition may provide a new therapeutic strategy for protecting both RGCs and their axons in glaucoma and other optic neuropathies.

Project description:Determining a representative microbial signature from any given location is dependent on robust sample collection and handling. Different sampling locations and hence sample properties can vary widely; for example, soil would be collected and handled differently compared to liquid samples. In the event that sample material has a low concentration of biomass, large quantities need to be collected for microbial community analysis. This is certainly the case when investigating the microbiology of oilfield systems, wherein produced water (PW) is one of the most common sources for microbial sampling. As the detrimental effects of microbial metabolism within these industrial milieus are becoming increasingly well-established, the characterization of microbial community composition using molecular biological analyses is becoming more commonplace for accurate monitoring. As this field continues to develop, the importance for standardized operating protocols cannot be understated, so that industry can make the most informed operational decisions possible. Accurately identifying oilfield microbial communities is paramount, as improper preservation and storage following sample collection is known to lead to erroneous microbial identifications. Preserving oilfield PW can be challenging, as many locations are remote, requiring lengthy periods of time before samples can be processed and analyzed. While previous studies have characterized the effects of various preservatives on concentrated, filtered, or purified microbial samples, to the best of our knowledge, no such study has been undertaken on low biomass liquid samples. To this end, we investigated the effectiveness of nine different preservation conditions on PW collected from the same sampling location within a heavy-oil producing field, and monitored how the microbial community changed over the period of a month. Our results reveal that the choice of preservative drastically affects microbial community, and should be selected with careful consideration before sampling occurs.

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Dataset Information

AAV expressing an mTOR-inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity.

Publications

AAV expressing an mTOR-inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity.

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