Small extracellular vesicles-transported lncRNA TDRKH-AS1 derived from AOPPs-treated trophoblasts initiates endothelial cells pyroptosis through PDIA4/DDIT4 axis in preeclampsia.

BACKGROUND: Substantial studies have demonstrated that oxidative stress placenta and endothelial injury are considered to inextricably critical events in the pathogenesis of preeclampsia (PE). Systemic inflammatory response and endothelial dysfunction are induced by the circulating factors released from oxidative stress placentae. As a novel biomarker of oxidative stress, advanced oxidation protein products (AOPPs) levels are strongly correlated with PE characteristics. Nevertheless, the molecular mechanism underlying the effect of factors is still largely unknown. METHODS: With the exponential knowledge on the importance of placenta-derived extracellular vesicles (pEVs), we carried out lncRNA transcriptome profiling on small EVs (sEVs) secreted from AOPPs-treated trophoblast cells and identified upregulated lncRNA TDRKH-AS1 as a potentially causative factor for PE. We isolated and characterized sEVs from plasma and trophoblast cells by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting. The expression and correlation of lncRNA TDRKH-AS1 were evaluated using qRT-PCR in plasmatic sEVs and placentae from patients. Pregnant mice injected with TDRKH-AS1-riched trophoblast sEVs was performed to detect the TDRKH-AS1 function in vivo. To investigate the potential effect of sEVs-derived TDRKH-AS1 on endothelial function in vitro, transcriptome sequencing, scanning electron Microscopy (SEM), immunofluorescence, ELISA and western blotting were conducted in HUVECs. RNA pulldown, mass spectrometry, RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP) and coimmunoprecipitation (Co-IP) were used to reveal the latent mechanism of TDRKH-AS1 on endothelial injury. RESULTS: The expression level of TDRKH-AS1 was significantly increased in plasmatic sEVs and placentae from patients, and elevated TDRKH-AS1 in plasmatic sEVs was positively correlated with clinical severity of the patients. Moreover, pregnant mice injected with TDRKH-AS1-riched trophoblast sEVs exhibited a hallmark feature of PE with increased blood pressure and systemic inflammatory responses. Pyroptosis, an inflammatory form of programmed cell death, is involved in the development of PE. Indeed, our in vitro study indicated that sEVs-derived TDRKH-AS1 secreted from AOPPs-induced trophoblast elevated DDIT4 expression levels to trigger inflammatory response of pyroptosis in endothelial cells through interacting with PDIA4. CONCLUSIONS: Herein, results in the present study supported that TDRKH-AS1 in sEVs isolated from oxidative stress trophoblast may be implicated in the pathogenesis of PE via inducing pyroptosis and aggravating endothelial dysfunction.

High-energy polarized soliton synthesis and its application to deep-brain 3-photon microscopy in vivo.

Here, we demonstrate a polarized high-energy soliton synthesis technique for deep-brain 3-photon microscopy (3PM) excited at the 1700-nm window. Through coherent combining, we generate linearly polarized high-energy solitons whose energy is twice as high than those of each linearly polarized solitons. Due to the nonlinear origin of signals, both measured 3-photon fluorescence signal and third-harmonic signals are thus boosted by ~8 times in a tissue phantom. Using this technique, we further demonstrate 3PM of sulforhodamine 101 labeled vasculature 1600 µm in the mouse brain in vivo, which cannot be achieved by single-polarized soliton excitation.

Order-of-magnitude multiphoton signal enhancement based on characterization of absorption spectra of immersion oils at the 1700-nm window.

To enhance signal levels in multiphoton microscopy (MPM) at the deep-tissue excitation window (1600-1820 nm) with oil immersion, we demonstrate: First, the absorption spectra of several commonly immersion oils are characterized, which were unknown before. Second, new material with lower absorption based on mixing is proposed. Third, optimal selection of excitation wavelength within this window is proposed based on absorption spectra characterization. Second and third harmonic generation imaging of mouse tissue corroborate our selection: 1600-nm excitation leads to notable orders-of-magnitude increase in MPM signal, compared with 1700-nm excitation, enabling 200-µm imaging depth of mouse skin while 1700-nm excitation could resolve virtually no structure.

Cyclosporin A alleviates trophoblast apoptosis and senescence by promoting autophagy in preeclampsia.

INTRODUCTION: Abnormal extravillous trophoblast (EVT) function is closely related to preeclampsia (PE) and may be caused by inadequate autophagy, apoptosis, and senescence. Cyclosporin A (CsA) is an effective immunosuppressant that has been reported to stimulate autophagy and exert benign biological effects on EVTs. Therefore, we hypothesized that CsA may display therapeutic efficacy against PE by activating autophagy. METHODS: We established the nitro-l-arginine methyl ester (l-NAME)-induced preeclamptic mice model and a hypoxia-reoxygenation (H/R) model in vitro. The effects of CsA on autophagy were evaluated by western blotting (WB). The effects of CsA on apoptosis were analyzed by Hematoxylin-eosin (H&E) staining, cell apoptosis assay and WB. Senescence-associated ß-galactosidase (SA-ß-gal) staining, RT-qPCR and WB were used to examine the senescence level. RT-qPCR were used to detect the senescence-associated secretory phenotype (SASP) level. DCFH-DA fluorescent probe, dihydroethidium (DHE) staining and mitochondrial membrane potential (ΔΨm) were used to detect senescence-associated mitochondrial dysfunction (SAMD). RESULTS: CsA alleviated PE-like symptoms and reduced placental necrosis and senescence in mice injected with l-NAME. CsA ameliorated placental SASP and SAMD level induced by l-NAME. CsA also upregulated the expression of autophagic proteins in mouse placentas disrupted using l-NAME. In vitro, we found that CsA reversed H/R-induced apoptosis and senescence, as well as decreasing SASP and SAMD levels and upregulating autophagic proteins levels. Notably, 3-methyladenine (3-MA), an early phase inhibitor of autophagosome formation, abolished the protective effects of CsA against H/R. DISCUSSION: CsA may display some therapeutic effects against PE by activating autophagy in vivo and in vitro.

Visualizing astrocytes in the deep mouse brain in vivo.

Astrocytes play a key role in the central nervous system. However, methods of visualizing astrocytes in the deep brain in vivo have been lacking. 3-photon fluorescence imaging of astrocytes labeled by sulforhodamine 101 (SR101) is demonstrated in deep mouse brain in vivo. Excitation wavelength selection was guided by wavelength-dependent 3-photon action cross section (ησ 3 ) measurement of SR101. 3-photon fluorescence imaging of the SR101-labeled vasculature enabled an imaging depth of 1340-µm into the mouse brain. This justifies the deep imaging capability of the technique and indicates that the imaging depth is not determined by the signal-to-background ratio limit encountered in 2-photon fluorescence imaging. Visualization of astrocytes 910 µm below the surface of the mouse brain in vivo is demonstrated, 30% deeper than that using 2-photon fluorescence microscopy. Through quantitative comparison of the signal difference between the SR101-labeled blood vessels and astrocytes, the challenges of visualizing astrocytes below the white matter is further elucidated.

Self-referenced axial chromatic dispersion measurement in multiphoton microscopy through 2-color third-harmonic generation imaging.

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2-color third-harmonic generation imaging excited by a 2-color soliton source with tunable wavelength separation. Our technique is self-referenced, eliminating potential measurement error when 1-color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2-color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.