Design, Control, and Experiments of a Novel Robotic Uterine Manipulator With the Motorized 3-DoF Manipulation Rod.

To address the issue of declining performance over time with manual uterine manipulation during minimally invasive gynecologic surgery, we propose a novel uterine manipulation robot that consists of a 3-DoF remote center of motion (RCM) mechanism and a 3-DoF manipulation rod. This allows for tireless, stable, and safer manipulation in place of a human assistant. For the RCM mechanism, we propose a single-motor bilinear-guided mechanism that can achieve a wide range of pitch motion (-50  âˆ¼  34 degrees) while maintaining a compact structure. This novel uterine manipulation robot is equipped with a manipulation rod that has a tip diameter of only 6 mm, allowing it to accommodate almost any patient's cervix. The 30-degree distal pitch motion and ±45-degree distal roll motion of the instrument further improve uterine visualization. Additionally, the tip of the rod can be opened into a T-shape to minimize damage to the uterus. Laboratory experiments have shown the mechanical RCM accuracy of 0.373 mm and the maximum load of the distal pitch joint of 500 g. Feasibility has been demonstrated through ex-vivo and cadaver tests, as well as clinical trials.

A Robotic System With Embedded Open Microfluidic Chip for Automatic Embryo Vitrification.

Embryo vitrification is a fundamental technology utilized in assisted reproduction and fertility preservation. Vitrification involves sequential loading and unloading of cryoprotectants (CPAs) with strict time control, and transferring the embryo in a minimum CPA droplet to the vitrification straw. However, manual operation still cannot effectively avoid embryo loss, and the existing automatic vitrification systems have insufficient system reliability, and operate differently from clinical vitrification protocol. Through collaboration with in vitro fertilization (IVF) clinics, we are in the process realizing a robotic system that can automatically conduct the embryo vitrification process, including the pretreatment with CPAs, transfer of embryo to the vitrification straw, and cryopreservation with liquid nitrogen ( LN2). An open microfluidic chip (OMC) was designed to accommodate the embryo during the automatic CPAs pretreatment process. The design of two chambers connected by a capillary gap facilitated solution exchange around the embryo, and simultaneously reduced the risk of embryo loss in the flow field. In accordance to the well-accepted procedure and medical devices in manual operation, we designed the entire vitrification protocol, as well as the robotic prototype. In a practical experiment using mouse embryos, our robotic system showed a 100 % success rate in transferring and vitrifying the embryos, achieved comparable embryo survival rates (90.9 % versus 94.4 %) and development rates (90.0 % versus 94.1 %), when compared with the manual group conducted by the senior embryologist. With this study, we aim to facilitate the standardization of clinical vitrification from manual operation to a more efficient and reliable automated process.

Anchor-guided online meta adaptation for fast one-Shot instrument segmentation from robotic surgical videos.

The scarcity of annotated surgical data in robot-assisted surgery (RAS) motivates prior works to borrow related domain knowledge to achieve promising segmentation results in surgical images by adaptation. For dense instrument tracking in a robotic surgical video, collecting one initial scene to specify target instruments (or parts of tools) is desirable and feasible during the preoperative preparation. In this paper, we study the challenging one-shot instrument segmentation for robotic surgical videos, in which only the first frame mask of each video is provided at test time, such that the pre-trained model (learned from easily accessible source) can adapt to the target instruments. Straightforward methods transfer the domain knowledge by fine-tuning the model on each given mask. Such one-shot optimization takes hundred of iterations and the test runtime is unfeasible. We present anchor-guided online meta adaptation (AOMA) for this problem. We achieve fast one-shot test time optimization by meta-learning a good model initialization and learning rates from source videos to avoid the laborious and handcrafted fine-tuning. The trainable two components are optimized in a video-specific task space with a matching-aware loss. Furthermore, we design an anchor-guided online adaptation to tackle the performance drop throughout a robotic surgical sequence. The model is continuously adapted on motion-insensitive pseudo-masks supported by anchor matching. AOMA achieves state-of-the-art results on two practical scenarios: (1) general videos to surgical videos, (2) public surgical videos to in-house surgical videos, while reducing the test runtime substantially.

Soft robotic manipulator for intraoperative MRI-guided transoral laser microsurgery.

Magnetic resonance (MR) imaging (MRI) provides compelling features for the guidance of interventional procedures, including high-contrast soft tissue imaging, detailed visualization of physiological changes, and thermometry. Laser-based tumor ablation stands to benefit greatly from MRI guidance because 3D resection margins alongside thermal distributions can be evaluated in real time to protect critical structures while ensuring adequate resection margins. However, few studies have investigated the use of projection-based lasers like those for transoral laser microsurgery, potentially because dexterous laser steering is required at the ablation site, raising substantial challenges in the confined MRI bore and its strong magnetic field. Here, we propose an MR-safe soft robotic system for MRI-guided transoral laser microsurgery. Owing to its miniature size (Ø12 × 100 mm), inherent compliance, and five degrees of freedom, the soft robot ensures zero electromagnetic interference with MRI and enables safe and dexterous operation within the confined oral and pharyngeal cavities. The laser manipulator is rapidly fabricated with hybrid soft and hard structures and is powered by microvolume (<0.004 milliter) fluid flow to enable laser steering with enhanced stiffness and lowered hysteresis. A learning-based controller accommodates the inherent nonlinear robot actuation, which was validated with laser path-following tests. Submillimeter laser steering accuracy was demonstrated with a mean error < 0.20 mm. MRI compatibility testing demonstrated zero observable image artifacts during robot operation. Ex vivo tissue ablation and a cadaveric head-and-neck trial were carried out under MRI, where we employed MR thermometry to monitor the tissue ablation margin and thermal diffusion intraoperatively.

Accurate instance segmentation of surgical instruments in robotic surgery: model refinement and cross-dataset evaluation.

PURPOSE: Automatic segmentation of surgical instruments in robot-assisted minimally invasive surgery plays a fundamental role in improving context awareness. In this work, we present an instance segmentation model based on refined Mask R-CNN for accurately segmenting the instruments as well as identifying their types. METHODS: We re-formulate the instrument segmentation task as an instance segmentation task. Then we optimize the Mask R-CNN with anchor optimization and improved Region Proposal Network for instrument segmentation. Moreover, we perform cross-dataset evaluation with different sampling strategies. RESULTS: We evaluate our model on a public dataset of the MICCAI 2017 Endoscopic Vision Challenge with two segmentation tasks, and both achieve new state-of-the-art performance. Besides, cross-dataset training improved the performance on both segmentation tasks compared with those tested on the public dataset. CONCLUSION: Results demonstrate the effectiveness of the proposed instance segmentation network for surgical instruments segmentation. Cross-dataset evaluation shows our instance segmentation model presents certain cross-dataset generalization capability, and cross-dataset training can significantly improve the segmentation performance. Our empirical study also provides guidance on how to allocate the annotation cost for surgeons while labelling a new dataset in practice.

Activation of dopaminergic VTA inputs to the mPFC ameliorates chronic stress-induced breast tumor progression.

AIMS: Chronic stress plays an important role in promoting the progression and migration of cancers. However, little is known of any direct impact on tumor progression related to the regulation of emotion-related circuitry. The aim of this study was to explore the neural-circuit mechanisms underlying stress-induced progression of cancers and the impact of emotion-related regulation of circuitry on tumor growth. METHODS: Optogenetic manipulation was applied to unpredictable chronic mild stress (UCMS)-treated mice bearing breast tumor cell. The stress-related hormones, tumor-related cytokines, the tyrosine hydroxylase (TH)-positive neurons and their fibers, dopamine receptor-positive cells, and anxiety level were measured using ELISA, immunohistochemical staining, fluorescence in situ hybridization, and behavioral test, respectively. RESULTS: By investigating breast cancer mouse models with a chronic mild stress model, optogenetic stimulation, and behavioral analysis, we show that chronic stress induced anxiety-like behavior in mice and increased serum concentration of norepinephrine and corticosterone, hormones closely related to stress and anxiety. Optogenetic activation of VTA TH terminals in the mPFC rescued anxiety-like behavior induced by chronic stress. Chronic stress resulted in marked progression of breast tumors, and repetitive optogenetic activation of VTA TH terminals in the mPFC significantly attenuated stress-induced progression of breast cancers and reduced serum concentration of norepinephrine and corticosterone. Furthermore, there was a positive correlation between serum norepinephrine or corticosterone concentration and tumor size. CONCLUSIONS: These findings indicate a positive role of an emotion regulation circuit on the progression of breast cancer and reveal a link between stress, emotion regulation, and the progression of breast cancers. Our findings provide new insights pertinent to therapeutic interventions in the treatment of breast cancers.

A collaborative robotic uterine positioning system for laparoscopic hysterectomy: Design and experiments.

BACKGROUND: Uterus manipulation is a lengthy and tedious task that is usually performed by a human assistant during laparoscopic hysterectomy. Note that the performance of the assistant may decrease with time. Moreover, under this approach, the primary surgeon does not have direct control over the uterus position. He/she can only verbally request the assistant to place it on a particular configuration. METHODS: A robotic system composed of a 3 degrees-of-freedom uterine positioner is developed to assist in changing configuration of the uterus during laparoscopic hysterectomy. The developed system has a remote centre of motion structure; independently controlling the uterus motion with one joint at the time is allowed. RESULTS: From the lab experiments, it is found that the robot shows better performance in retaining the uterus position and shows quicker response to the surgeon's instruction. Cadaver studies have been conducted to evaluate the feasibility of the robot. The robot was also applied to real patients in a clinical study. CONCLUSIONS: The robot is capable of assisting in uterus manipulation during laparoscopic hysterectomy. However, its user friendliness can be improved by simplifying the docking procedure. Furthermore, a more ergonomic user interface is desired.

A Real-Time 3D Laparoscopic Imaging System: Design, Method, and Validation.

OBJECTIVE: This paper aims to propose a 3D laparoscopic imaging system that can realize dense 3D reconstruction in real time. METHODS: Based on the active stereo technique which yields high-density, accurate and robust 3D reconstruction by combining structured light and stereo vision, we design a laparoscopic system consisting of two image feedback channels and one pattern projection channel. Remote high-speed image acquisition and pattern generation lay the foundation for the real-time dense 3D surface reconstruction and enable the miniaturization of the laparoscopic probe. To enhance the reconstruction efficiency and accuracy, we propose a novel active stereo method by which the dense 3D point cloud is obtained using only five patterns, while most existing multiple-shot structured light techniques require [Formula: see text] patterns. In our method, dual-frequency phase-shifting fringes are utilized to uniquely encode the pixels of the measured targets, and a dual-codeword matching scheme is developed to simplify the matching procedure and achieve high-precision reconstruction. RESULTS: Compared with the existing structured light techniques, the proposed method shows better real-time efficiency and accuracy in both quantitative and qualitative ways. Ex-vivo experiments demonstrate the robustness of the proposed method to different biological organs and the effectiveness to lesions and deformations of the organs. Feasibility of the proposed system for real-time dense 3D reconstruction is verified in dynamic experiments. According to the experimental results, the system acquires 3D point clouds with a speed of 12 frames per second. Each frame contains more than 40,000 points, and the average errors tested on standard objects are less than 0.2 mm. SIGNIFICANCE: This paper provides a new real-time dense 3D reconstruction method for 3D laparoscopic imaging. The established prototype system has shown good performance in reconstructing surface of biological tissues.

Force-based Safe Vein Cannulation in Robot-assisted Retinal Surgery: A Preliminary Study.

Retinal vein cannulation (RVC) is a potential treatment for retinal vein occlusion (RVO). Manual surgery has limitations in RVC due to extremely small vessels and instruments involved, as well as the presence of physiological hand tremor. Robot-assisted retinal surgery may be a better approach to smooth and accurate instrument manipulation during this procedure. Motion of the retina and cornea related to heartbeat may be associated with unexpected forces between the tool and eyeball. In this paper, we propose a force-based control strategy to automatically compensate for the movement of the retina maintaining the tip force and sclera force in a predetermined small range. A dual force-sensing tool is used to monitor the tip force, sclera force and tool insertion depth, which will be used to derive a desired joint velocity for the robot via a modified admittance controller. Then the tool is manipulated to compensate for the movement of the retina as well as reduce the tip force and sclera force. Quantitative experiments are conducted to verify the efficacy of the control strategy and a user study is also conducted by a retinal surgeon to demonstrate the advantages of our automatic compensation approach.

An Optimized Tilt Mechanism for a New Steady-Hand Eye Robot.

Robot-assisted vitreoretinal surgery can filter surgeons' hand tremors and provide safe, accurate tool manipulation. In this paper, we report the design, optimization, and evaluation of a novel tilt mechanism for a new Steady-Hand Eye Robot (SHER). The new tilt mechanism features a four-bar linkage design and has a compact structure. Its kinematic configuration is optimized to minimize the required linear range of motion (LRM) for implementing a virtual remote center-of-motion (V-RCM) while tilting a surgical tool. Due to the different optimization constraints for the robots at the left and right sides of the human head, two configurations of this tilt mechanism are proposed. Experimental results show that the optimized tilt mechanism requires a significantly smaller LRM (e.g. 5.08 mm along Z direction and 8.77 mm along Y direction for left side robot) as compared to the slider-crank tilt mechanism used in the previous SHER (32.39 mm along Z direction and 21.10 mm along Y direction). The feasibility of the proposed tilt mechanism is verified in a mock bilateral robot-assisted vitreoretinal surgery. The ergonomically acceptable robot postures needed to access the surgical field is also determined.

Synergistic antitumor effect on cervical cancer by rational combination of PD1 blockade and CRISPR-Cas9-mediated HPV knockout.

Targeted therapy results in objective responses in cervical cancer. However, the responses are short. In contrast, treatment with immune checkpoint inhibitors results in a lower responses rate, but the responses tend to be more durable. Based on these findings, we hypothesized that HPV16 E6/E7-targeted therapy may synergize with the PD-1 pathway blockade to enhance antitumor activity. To test hypothesis, we described for the first time the effects of the CRISPR/Cas9 that was targeted to the HPV and PD1 in vitro and in vivo. Our data showed that gRNA/cas9 targeted HPV16 E6/E7 induced cervical cancer cell SiHa apoptosis, and suggested that overexpression of PD-L1, induced by HPV16 E6/E7, may be responsible for lymphocyte dysfunction. In established SiHa cell- xenografted humanized SCID mice, Administration of gRNA-PD-1 together with gRNA-HPV16 E6/E7 treatment improved the survival and suppressed the tumor growth obviously. In addition, combination treatment increased the population of dendritic cells, CD8+ and CD4+ T lymphocyte cells. According, it enhanced the expression of Th1-associated immune-stimulating genes while reducing the transcription of regulatory/suppressive immune genes, reshaping tumor microenvironment from an immunosuppressive to a stimulatory state. These results demonstrate potent synergistic effects of combination therapy using HPV16 E6/E7-targeted therapy and immune checkpoint blockade PD1, supporting a direct translation of this combination strategy in clinic for the treatment of cervical cancer.

Foot-Controlled Robot-Enabled EnDOscope Manipulator (FREEDOM) for Sinus Surgery: Design, Control, and Evaluation.

Despite successful clinical applications, teleoperated robotic surgical systems face particular limitations in the functional endoscopic sinus surgery (FESS) in terms of incompatible instrument dimensions and robot set-up. The endoscope remains manually handled by an assistant when the surgeon performs bimanual operations. This paper introduces the development of the Foot-controlled Robot-Enabled EnDOscope Manipulator (FREEDOM) designed for FESS. The system features clinical considerations that inform the design for providing reliable and safe endoscope positioning with minimal obstruction to the routine practice. The robot structure is modular and compact to ensure coaxial instrument manipulation through the nostril for manual procedures. To avoid rigid endoscope motions, a new compliant endoscope holder is proposed that passively limits the lens-tissue contact forces under collisions for patient-side protection. To facilitate hands-free endoscope manipulation that imposes minimal distractions to the surgeon, a foot-wearable interface is further designed to relieve the assistant's workload. The foot control method owns a short learning curve (mean 3.4 mins), and leads the task to be more ergonomic and surgeon-centered. Cadaver and clinical studies were both conducted to evaluate the surgical applicability of the FREEDOM to assist endoscope manipulation in FESS. The system was validated to be safe (IEC-60601-1) and easy for set up (mean 3.6 mins), from which the surgeon could perform various three-handed procedures alone in FESS without disrupting the routine practice.

WITHDRAWN: CRISPR/Cas9 mediated HPV and PD1 inhibition produces a synergistic anti-tumor effect on cervical cancer.

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Toward Semi-autonomous Cryoablation of Kidney Tumors via Model-Independent Deformable Tissue Manipulation Technique.

We present a novel semi-autonomous clinician-in-the-loop strategy to perform the laparoscopic cryoablation of small kidney tumors. To this end, we introduce a model-independent bimanual tissue manipulation technique. In this method, instead of controlling the robot, which inserts and steers the needle in the deformable tissue (DT), the cryoprobe is introduced to the tissue after accurate manipulation of a target point on the DT to the desired predefined insertion location of the probe. This technique can potentially reduce the risk of kidney fracture, which occurs due to the incorrect insertion of the probe within the kidney. The main challenge of this technique, however, is the unknown deformation behavior of the tissue during its manipulation. To tackle this issue, we proposed a novel real-time deformation estimation method and a vision-based optimization framework, which do not require prior knowledge about the tissue deformation and the intrinsic/extrinsic parameters of the vision system. To evaluate the performance of the proposed method using the da Vinci Research Kit, we performed experiments on a deformable phantom and an ex vivo lamb kidney and evaluated our method using novel manipulability measures. Experiments demonstrated successful real-time estimation of the deformation behavior of these DTs while manipulating them to the desired insertion location(s).

Inhibition of long non-coding RNA UCA1 by CRISPR/Cas9 attenuated malignant phenotypes of bladder cancer.

CRISPR/Cas9 is a novel and effective genome editing technique, but its application is not widely expanded to manipulate long non-coding RNA (lncRNA) expression. The lncRNA urothelial carcinoma-associated 1 (UCA1) is upregulated in bladder cancer and promotes the progression of bladder cancer. Here, we design gRNAs specific to UCA1 and construct CRISPR/Cas9 systems targeting UCA1. Single CRISPR/Cas9-UCA1 can effectively inhibit UCA1 expression when transfected into 5637 and T24 bladder cancer cells, while the combined transfection of the two most effective CRISPR/Cas9-UCA1s can generate more satisfied inhibitory effect. CRISPR/Cas9-UCA1s attenuate UCA1 expression via targeted genome-specific DNA cleavage, resulting in the significant inhibition of cell proliferation, migration and invasion in vitro and in vivo. The mechanisms associated with the inhibitory effect of CRISPR/Cas9-UCA1 on malignant phenotypes of bladder cancer are attributed to the induction of cell cycle arrest at G1 phase, a substantial increase of apoptosis, and an enhanced activity of MMPs. Additionally, urinary UCA1 can be used as a non-invasive diagnostic marker for bladder cancer as revealed by a meta-analysis. Collectively, our data suggest that CRISPR/Cas9 technique can be used to down-modulate lncRNA expression, and urinary UCA1 may be used as a non-invasive marker for diagnosis of bladder cancer.

MiR-383 inhibits proliferation, migration and angiogenesis of glioma-exposed endothelial cells in vitro via VEGF-mediated FAK and Src signaling pathways.

Malignant glioma is undoubtedly the most vascularized tumor of central nervous system. Angiogenesis, playing a predominant role in tumor progression, is widely considered as a key point of tumor treatment. The aim of this study was to investigate the potential effects of miR-383 on proliferation, migration, tube formation and angiogenesis of glioma-exposed endothelial cells (GECs) in vitro and to further elucidate its possible molecular mechanisms. The expression of miR-383 in GECs was significantly downregulated compared with that in normal endothelial cells (ECs). Overexpression of miR-383 dramatically inhibited the proliferation, migration, tube formation and spheroid-based angiogenesis of GECs in vitro. Dual-luciferase reporter results demonstrated vascular endothelial growth factor (VEGF) is a target gene of miR-383. Furthermore, overexpression or silencing of either miR-383 or VEGF was performed simultaneously to further clarify that miR-383 inhibited proliferation, migration and angiogenesis of GECs in vitro by targeting VEGF. Finally, VEGF/VEGFR2-mediated FAK and Src signaling pathways might contribute to anti-angiogenesis of GECs. In conclusion, our present study indicated that miR-383 inhibits proliferation, migration and angiogenesis of GECs in vitro via VEGF/VEGFR2-mediated FAK and Src signaling pathways, which would draw growing attention to miR-383c as a potential therapeutical target of glioma.

miR-128 regulates the apoptosis and proliferation of glioma cells by targeting RhoE.

In this study, we investigate whether miR-128 is capable of regulating the apoptosis and proliferation of human U251 glioma cells by downregulating RhoE. The expression of miR-128 was assessed by quantitative polymerase chain reaction in normal brain tissue and glioma samples. A significant downregulation of the expression of miR-128 was detected in glioma in contrast to normal brain tissue. Following the transfection of pre-miR-128 and anti-miR-128 into U251 cells, the high expression of miR-128 could inhibit proliferation and induce apoptosis in U251 cells, and those effects could be restored by miR-128 knockdown. To analyze the regulation mechanism of miR-128, TargetScan, miRanda and PicTar were used to ascertain whether RhoE was a potential target gene. Next, luciferase activity assay and western blot analysis confirmed that RhoE was a direct and specific target gene of miR-128. The advanced effects of pre-miR-128 on the apoptosis and proliferation of U251 cells were reversed by the upregulation of RhoE expression. In summary, aberrantly expressed miR-128 regulates apoptosis and proliferation in human glioma U251 cells partly by directly targeting RhoE. This finding may offer a new potential therapeutic strategy for the treatment of glioma.

DT(270-326) , a Truncated Diphtheria Toxin, Increases Blood-Tumor Barrier Permeability by Upregulating the Expression of Caveolin-1.

AIM: The nontoxic mutant of diphtheria toxin (DT) has been demonstrated to act as a receptor-specific carrier protein to delivery drug into brain. Recent research showed that the truncated "receptorless" DT was still capable of being internalized into cells. This study investigated the effects and potential mechanisms of DT(270-326) , a truncated "receptorless" DT, on the permeability of the blood-tumor barrier (BTB). METHODS: BTB and GECs were subjected to DT(270-326) treatment. HRP flux assays, immunofluorescent, co-immunoprecipitation, Western blot, CCK-8, and Flow cytometry analysis were used to evaluate the effects of DT(270-326) administration. RESULTS: Our results revealed that 5 µM of DT(270-326) significantly increased the permeability of BTBin vitro, which reached its peak at 6 h. The permeability was reduced by pretreatment with filipinIII. DT(270-326) co-localized and interacted with caveolin-1 via its caveolin-binding motif. The mRNA and protein expression levels of caveolin-1 were identical with the changes of BTB permeability. The upregulated expression of caveolin-1 was associated with Src kinase-dependent tyrosine phosphorylation of caveolin-1, which subsequently induced phosphorylation and inactivation of the transcription factor Egr-1. The combination of DT(270-326) with doxorubicin significantly enhanced the loss of cell viability and apoptosis of U87 glioma cells in contrast to doxorubicin alone. CONCLUSIONS: DT(270-326) might provide a novel strategy to increase the delivery of macromolecular therapeutic agents across the BTB.

Foot-controlled robotic-enabled endoscope holder for endoscopic sinus surgery: A cadaveric feasibility study.

OBJECTIVES/HYPOTHESIS: To evaluate the feasibility of a unique prototype foot-controlled robotic-enabled endoscope holder (FREE) in functional endoscopic sinus surgery. STUDY DESIGN: Cadaveric study. METHODS: Using human cadavers, we investigated the feasibility, advantages, and disadvantages of the robotic endoscope holder in performing endoscopic sinus surgery with two hands in five cadaver heads, mimicking a single nostril three-handed technique. RESULTS: The FREE robot is relatively easy to use. Setup was quick, taking less than 3 minutes from docking the robot at the head of the bed to visualizing the middle meatus. The unit is also relatively small, takes up little space, and currently has four degrees of freedom. The learning curve for using the foot control was short. The use of both hands was not hindered by the presence of the endoscope in the nasal cavity. The tremor filtration also aided in the smooth movement of the endoscope, with minimal collisions. CONCLUSION: The FREE endoscope holder in an ex-vivo cadaver test corroborated the feasibility of the robotic prototype, which allows for a two-handed approach to surgery equal to a single nostril three-handed technique without the holder that may reduce operating time. Further studies will be needed to evaluate its safety profile and use in other areas of endoscopic surgery. LEVEL OF EVIDENCE: NA. Laryngoscope, 126:566-569, 2016.

Low-Dose Endothelial Monocyte-Activating Polypeptide-II Induces Blood-Tumor Barrier Opening Via the cAMP/PKA/Rac1 Pathway.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent signaling pathway is involved in EMAP-II-induced BTB hyperpermeability. This study further investigated the exact mechanisms through which the cAMP/PKA-dependent signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant decrease in Rac1 activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced inactivation of Rac1. Besides, pretreatment with 6Bnz-cAMP to activate PKA partially attenuated EMAP-II-induced Rac1 inactivation. Moreover, 6Bnz-cAMP pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, myosin light chain (MLC) phosphorylation, expression and distribution of ZO-1, and actin cytoskeleton arrangement in RBMECs. These effects of 6Bnz-cAMP were completely blocked in the presence of NSC-23766 (the specific inhibitor of Rac1). In conclusion, this study demonstrates that low-dose EMAP-II induces BTB hyperpermeability via the cAMP/PKA/Rac1 signaling pathway.