Anti-Proliferation Effect of Theasaponin E1 on the ALDH-Positive Ovarian Cancer Stem-Like Cells.

Ovarian cancer has the highest mortality rate of all gynecological malignancies and the five-year death rate of patients has remained high in the past five decades. Recently, with the rise of cancer stem cells (CSCs) theory, an increasing amount of research has suggested that CSCs give rise to tumor recurrence and metastasis. Theasaponin E1 (TSE1), which was isolated from green tea (Camellia sinensis) seeds, has been proposed to be an effective compound for tumor treatment. However, studies on whether TSE1 takes effect through CSCs have rarely been reported. In this paper, ALDH-positive (ALDH+) ovarian cancer stem-like cells from two platinum-resistant ovarian cancer cell lines A2780/CP70 and OVCAR-3 were used to study the anti-proliferation effect of TSE1 on CSCs. The ALDH+ cells showed significantly stronger sphere forming vitality and stronger cell migration capability. In addition, the stemness marker proteins CD44, Oct-4, Nanog, as well as Bcl-2 and MMP-9 expression levels of ALDH+ cells were upregulated compared with the original tumor cells, indicating that they have certain stem cell characteristics. At the same time, the results showed that TSE1 could inhibit cell proliferation and suspension sphere formation in ALDH+ cells. Our data suggests that TSE1 as a natural compound has the potential to reduce human ovarian cancer mortality. However, more research is still needed to find out the molecular mechanism of TSE1-mediated inhibition of ALDH+ cells and possible drug applications on the disease.

Reactive oxygen species-caspase-3 relationship in mediating blood-brain barrier endothelial cell hyperpermeability following oxygen-glucose deprivation and reoxygenation.

OBJECTIVE: Microvascular hyperpermeability that occurs due to breakdown of the BBB is a major contributor of brain vasogenic edema, following IR injury. In microvascular endothelial cells, increased ROS formation leads to caspase-3 activation following IR injury. The specific mechanisms, by which ROS mediates microvascular hyperpermeability following IR, are not clearly known. We utilized an OGD-R in vitro model of IR injury to study this. METHODS: RBMEC were subjected to OGD-R in presence of a caspase-3 inhibitor Z-DEVD, caspase-3 siRNA or an ROS inhibitor L-AA. Cytochrome c levels were measured by ELISA and caspase-3 activity was measured fluorometrically. TJ integrity and cytoskeletal assembly were studied using ZO-1 immunofluorescence and rhodamine phalloidin staining for f-actin, respectively. RESULTS: OGD-R significantly increased monolayer permeability, ROS formation, cytochrome c levels, and caspase-3 activity (p < 0.05) and induced TJ disruption and actin stress fiber formation. Z-DEVD, L-AA and caspase-3 siRNA significantly attenuated OGD-R-induced hyperpermeability (p < 0.05) while only L-AA decreased cytochrome c levels. Z-DEVD and L-AA protected TJ integrity and actin cytoskeletal assembly. CONCLUSIONS: These results suggest that OGD-R-induced hyperpermeability is ROS and caspase-3 dependent and can be regulated by their inhibitors.

Regulation of tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability by recombinant B-cell lymphoma-extra large.

BACKGROUND: Tumor necrosis factor-α (TNF-α), a cytotoxic cytokine, induces endothelial cell barrier dysfunction and microvascular hyperpermeability, leading to tissue edema, a hallmark of traumatic injuries. The objective of the present study was to determine whether B-cell lymphoma-extra large (Bcl-xL), an antiapoptotic protein, would regulate and protect against TNF-α-mediated endothelial cell barrier dysfunction and microvascular hyperpermeability. METHODS: Rat lung microvascular endothelial cells were grown as monolayers on Transwell membranes, and fluorescein isothiocyanate-bovine albumin flux (5 mg/mL) across the monolayer was measured fluorometrically to indicate changes in monolayer permeability. The rat lung microvascular endothelial cell adherens junctional integrity and actin cytoskeleton was studied using ß-catenin immunofluorescence and rhodamine phalloidin dye, respectively. Pretreatment of caspase-8 inhibitor (Z-IETD-FMK, 100 µM) for 1 hour and transfection of Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA (10 µM) for 48 hours were performed to study their respective effects on TNF-α-induced (10 ng/mL; 1-hour treatment) monolayer permeability. Recombinant Bcl-xL protein (2.5 µg/ml) was transfected in rat lung microvascular endothelial cells for 1 hour, and its effect on permeability was demonstrated using a permeability assay. Caspase-3 activity was assayed fluorometrically. RESULTS: Z-IETD-FMK pretreatment protected the adherens junctions and decreased TNF-α-induced monolayer hyperpermeability. Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA transfection attenuated the TNF-α-induced increase in monolayer permeability. Recombinant Bcl-xL protein showed protection against TNF-α-induced actin stress fiber formation, an increase in caspase-3 activity, and monolayer hyperpermeability. CONCLUSIONS: Our results have demonstrated the protective effects of recombinant Bcl-xL protein against TNF-α-induced endothelial cell adherens junction damage and microvascular endothelial cell hyperpermeability. These findings support the potential for Bcl-xL-based drug development against microvascular hyperpermeability and tissue edema.

Prolonged generalized immune response on 18F-FDG PET/CT following COVID-19 vaccination.

The Coronavirus disease 2019 (COVID-19) pandemic continues to be a major public health concern affecting millions of people globally. The COVID-19 vaccination has implications in medical assessment of cancer patients especially undergoing diagnostic imaging such as 18F-fluoro-deoxyglucose (FDG) positron emission tomography with computed tomography (PET/CT). The inflammatory changes following vaccination can cause false positive findings on imaging. We present a case of a patient with esophageal carcinoma who had 18F-FDG PET/CT scan, 8 weeks following booster dose of Moderna COVID-19 vaccination, which showed widespread FDG avid reactive lymph nodes and intense splenic uptake for prolonged duration of approximately 8 months (34 weeks) probably representing generalized immune response. It is important from radiological/nuclear medicine perspective to recognize imaging features of such rare effect of COVID-19 vaccination, which can pose a challenge in assessing 18F-FDG PET/CT scans in cancer patients. It has also opened new avenues for future research evaluating such COVID-19 vaccine-related prolonged systemic immunological response in cancer patients.

Inhibition of VE-cadherin proteasomal degradation attenuates microvascular hyperpermeability.

OBJECTIVE: VE-cadherin, an integral component of the adherens junction complex, is processed through the endosome-lysosome pathway and proteasome system for degradation. Our objective was to determine if inhibition of this pathway would protect against microvascular hyperpermeability. METHODS: To induce VE-cadherin degradation, we utilized a mutant VE-cadherin protein that lacks the extracellular domain (rVE-cad CPD). Intravital microscopy was employed to study the changes in microvascular permeability in rat mesenteric postcapillary venules. Rat lung microvascular endothelial cell (RLMEC) monolayers were utilized in parallel studies. The adherens junction integrity was determined using VE-cadherin and ß-catenin immunofluorescence. TOPflash/FOPflash transfection and luciferase reporter assay were performed to study ß-catenin-mediated transcriptional activation. RESULTS: rVE-cad CPD (2.5 µg/mL of blood volume) increased hyperpermeability significantly (p < 0.05). The VE-cadherin siRNA as well as rVE-cad CPD induced significant increase in monolayer hyperpermeability (p < 0.05). Transfection of rVE-cad CPD disrupted adherens junctions evidenced by discontinuity in ß-catenin and VE-cadherin immunofluorescence (p < 0.05). Proteasome inhibitor MG132 attenuated rVE-cad CPD induced monolayer hyperpermeability and adherens junction damage. CONCLUSIONS: VE-cadherin disruption in animals results in hyperpermeability. Parallel studies in RLMEC demonstrated similar results. In addition, inhibition of proteasomal degradation attenuated microvascular hyperpermeability. These findings have significance in understanding the role of VE-cadherin in regulating vascular hyperpermeability.

Role of ß-catenin in regulating microvascular endothelial cell hyperpermeability.

BACKGROUND: Paracellular microvascular hyperpermeability occurs mainly because of the disruption of the endothelial adherens junction complex. Vascular endothelial-cadherin that consists of an extracellular and intracellular domain to confer cell-cell contact is linked to the actin cytoskeletal assembly through ß-catenin. Our objective was to determine the functional role of ß-catenin during paracellular hyperpermeability and to evaluate whether exogenous ß-catenin would protect against vascular leak. METHODS: ß-Catenin siRNA (2.5 µg/mL) was administered to Sprague-Dawley rats through tail vein. FITC-albumin extravasation of the mesenteric postcapillary venules was evaluated after 48 hours using intravital microscopy. Parallel studies using rat lung microvascular endothelial cell monolayers were transfected with ß-catenin siRNA, and hyperpermeability was determined using monolayers after 48 hours. The effectiveness of ß-catenin siRNA was tested using immunofluorescence and Western blot. To study the protective effect of ß-catenin, rat lung microvascular endothelial cell monolayers were transfected with a ß-catenin gene expression construct for 48 hours or a recombinant ß-catenin protein (1 µg/mL) for 2 hours, followed by transfection with proapoptotic BAK peptide (5 µg/mL), a known inducer hyperpermeability. RESULTS: ß-Catenin siRNA induced a significant increase in vascular hyperpermeability in vivo (p<0.05) and monolayer permeability (in vitro; p<0.05). ß-Catenin siRNA significantly altered the adherens junction complex and decreased ß-catenin protein levels. ß-Catenin gene expression construct or recombinant ß-catenin protein attenuated BAK-induced monolayer hyperpermeability significantly (p<0.05). CONCLUSION: Posttranscriptional gene silencing of ß-catenin leads to vascular hyperpermeability in vivo and monolayer hyperpermeability in vitro. The enhancement of ß-catenin gene expression at the adherens junction or exogenous introduction of ß-catenin protein shows protection against vascular hyperpermeability.

An Unusual Course of Segmental Renal Artery Displays a Rare Case of Hilar Nutcracker Phenomenon.

Nutcracker phenomenon or renal vein entrapment is classically seen as a compression of renal vein in between abdominal aorta and superior mesenteric artery with patients being asymptomatic or clinically manifested in the form of nutcracker syndrome as proteinuria, hematuria, flank pain, pelvic congestion in women, and varicocele in men. In this report, we are presenting a case of rare variant of nutcracker phenomenon along with brief review of anatomy, pathophysiology, public health, and clinical significance of nutcracker syndrome. On a routine dissection of an adult male cadaver, we noticed an unusual arrangement of the structures at the hilum of the left kidney showing entrapment of renal vein between left anterior inferior and posterior segmental renal arteries. The variation in the course of left anterior inferior segmental renal artery leads to compression of left renal vein at renal hilum. Therefore, we have named this rare abnormal anatomical entity as hilar nutcracker phenomenon. The structures in the right renal hilum are normal. The objective of this paper is to report an unusual but important variant of nutcracker phenomenon and also give collective knowledge of such anatomical variations in renal vasculature that will help in diagnosing and treating such rare renal disorder.

Glycogen synthase kinase 3 inhibitor protects against microvascular hyperpermeability following hemorrhagic shock.

BACKGROUND: Hemorrhagic shock (HS)-induced microvascular hyperpermeability involves disruption of endothelial cell adherens junctions leading to increase in paracellular permeability. ß-Catenin, an integral component of the adherens junctional complex and Wnt pathway, and caspase 3 via its apoptotic signaling regulate endothelial cell barrier integrity. We have hypothesized that inhibiting phosphorylation of ß-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 would attenuate microvascular hyperpermeability following HS. METHODS: In Sprague-Dawley rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mm Hg for 60 minutes followed by resuscitation. Rats were given SB216763 (600 µg/kg) intravenously 10 minutes before shock. To study microvascular permeability, the rats were intravenously injected with fluorescein isothiocyanate (FITC)-albumin (50 mg/kg), and its flux across the mesenteric postcapillary venules was determined using intravital microscopy. In cell culture studies, rat lung microvascular endothelial cell monolayers grown on Transwell plates were pretreated with SB216763 (5 µM) followed by BAK (5 µg/mL) and caspase 3 (5 µg/mL) protein transfection. FITC-albumin (5 mg/mL) flux across cell monolayers indicates change in monolayer permeability. Activity of canonical Wnt pathway was determined by luciferase assay. Caspase 3 enzyme activity was assayed fluorometrically. RESULTS: The HS group showed significant increase in FITC-albumin extravasation (p < 0.05) compared with sham. SB216763 significantly decrease HS-induced FITC-albumin extravasation (p < 0.05). Pretreatment with SB216763 protected against a BAK-induced increase in rat lung microvascular endothelial cell monolayer permeability and caspase 3 activity but failed to show similar results with a caspase 3-induced increase in monolayer permeability. Wnt3a treatment showed an increase in ß-catenin-dependent T-cell factor-mediated transcription. CONCLUSION: Inhibiting phosphorylation of ß-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 help regulates HS-induced microvascular hyperpermeability.

RECOMBINANT BCL-XL ATTENUATES VASCULAR HYPERPERMEABILITY IN A RAT MODEL OF HEMORRHAGIC SHOCK.

Following hemorrhagic shock (HS), vascular hyperpermeability i.e. the leakage of fluid, nutrients and proteins into the extravascular space occurs primarily due to the disruption of the endothelial cell-cell adherens junctional complex. Studies from our laboratory demonstrate that activation of the mitochondria mediated 'intrinsic' apoptotic signaling cascade has a significant role in modulating HS-induced hyperpermeability. Here we report the novel use of recombinant Bcl-xL, an anti-apoptotic protein, to control HS-induced vascular hyperpermeability. Our results corroborate involvement of vascular hyperpermeability and apoptotic signaling. Hemorrhagic shock (HS) (mean arterial pressure [MAP] was reduced to 40 mmHg for 60 minutes followed by resuscitation to 90 mmHg for 60 minutes) in rats resulted in vascular hyperpermeability as determined by intra-vital microscopy. Treatment of Bcl-xL (2.5ug/ml of rat blood in non-lipid cationic polymer, i.v.) before, during and even after HS attenuated or reversed HS-induced vascular hyperpermeability significantly (p<0.05). Conversely, treatment using Bcl-xL inhibitors, 2-methoxy antimycin (2-MeOAA) and ABT 737, significantly increased vascular hyperpermeability compared to sham (p<0.05). Bcl-xL treatment also decreased the amount of fluid volume required to maintain a MAP of 90 mmHg during resuscitation (p<0.05). HS resulted in increased mitochondrial ROS formation, reduction of ΔΨm, mitochondrial release of cytochrome c and significant activation of caspase-3 (p<0.05). All of these effects were significantly inhibited by Bcl-xL pre-treatment (p<0.05). Our results show that recombinant Bcl-xL is effective against HS-induced vascular hyperpermeability that appears to be mediated through preservation of ΔΨm and subsequent prevention of caspase-3 activation.

The role of intrinsic apoptotic signaling in hemorrhagic shock-induced microvascular endothelial cell barrier dysfunction.

Hemorrhagic shock leads to endothelial cell barrier dysfunction resulting in microvascular hyperpermeability. Hemorrhagic shock-induced microvascular hyperpermeability is associated with worse clinical outcomes in patients with traumatic injuries. The results from our laboratory have illustrated a possible pathophysiological mechanism showing involvement of mitochondria-mediated "intrinsic" apoptotic signaling in regulating hemorrhagic shock-induced microvascular hyperpermeability. Hemorrhagic shock results in overexpression of Bcl-2 family of pro-apoptotic protein, BAK, in the microvascular endothelial cells. The increase in BAK initiates "intrinsic" apoptotic signaling cascade with the release of mitochondrial cytochrome c in the cytoplasm and activation of downstream effector caspase-3, leading to loss of endothelial cell barrier integrity. Thus, this review article offers a brief overview of important findings from our past and present research work along with new leads for future research. The summary of our research work will provide information leading to different avenues in developing novel strategies against microvascular hyperpermeability following hemorrhagic shock.

Tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling.

Tumor necrosis factor-α (TNF-α), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-α induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-α (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 µM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using ß-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 µM) attenuated TNF-α-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-α (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability.

Tumor necrosis factor-related apoptosis-inducing ligand promotes microvascular endothelial cell hyperpermeability through phosphatidylinositol 3-kinase pathway.

BACKGROUND: Microvascular hyperpermeability that occurs in hemorrhagic shock and burn trauma is regulated by the apoptotic signaling pathway. We hypothesized that tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL) would promote hyperpermeability directly or by interacting with other signaling pathways. METHODS: Rat lung microvascular endothelial cells (RLMECs) grown on Transwell membranes (Corning Life Sciences, Lowell, MA) were treated with recombinant human TRAIL (10, 50, and 100 ng/mL) for 6 hours or TRAIL (100 ng/mL) + LY294002 (a PI3K inhibitor; 20 µmol/L), Z-DEVD-FMK (a caspase-3 inhibitor; 10 µmol/L), or the inhibitors alone. Fluorescein isothiocyanate (FITC)-albumin flux was an indicator of permeability. Caspase-3 activity was measured fluorometrically. Adherens junction integrity was studied using ß-catenin immunofluorescence. RESULTS: TRAIL + LY294002, but not TRAIL alone, induced monolayer hyperpermeability (P < .05), and caspase-3 activity (P < .05), and disrupted the adherens junctions. Z-DEVD-FMK attenuated hyperpermeability and protected the adherens junctions. CONCLUSIONS: TRAIL-induced microvascular hyperpermeability is phosphatidylinositol 3-kinase (PI3K)-dependent and may be mediated by caspase-3 cleavage of the endothelial adherens junctional complex.

Microvascular endothelial cell hyperpermeability induced by endogenous caspase 3 activator staurosporine.

BACKGROUND: Microvascular hyperpermeability following conditions such as hemorrhagic shock occurs mainly owing to disruption of the adherens junctional protein complex in endothelial cells. The objective of this study was to examine the action of staurosporine, a potent activator of endogenous caspase 3 on the adherens junction and the cellular pathway through which it causes possible endothelial cell barrier dysfunction. METHODS: Rat lung microvascular endothelial cell (RLMEC) permeability was measured by fluorescein isothiocyanate-albumin flux across the monolayer in a Transwell plate. Integrity of the endothelial cell adherens junctions was studied using immunofluorescence of ß-catenin and vascular endothelial-cadherin. Mitochondrial reactive oxygen species formation was determined by using dihydrorhodamine 123 and mitochondrial transmembrane potential by JC-1 fluorescent probe and flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Cell death assay in RLMECs was performed using propidium iodide staining and analyzed by flow cytometry. RESULTS: Staurosporine (1 µM)-treated RLMEC monolayers showed significant increase in permeability, which was decreased by pretreatment with caspase 3 specific inhibitor, Z-DEVD-FMK (p < 0.05). Immunofluorescence studies showed staurosporine induced disruption of the adherens junction, which was reversed by Z-DEVD-FMK. Staurosporine treatment led to an increase in mitochondrial reactive oxygen species formation and a decrease in mitochondrial transmembrane potential. Furthermore, staurosporine induced a significant increase in caspase 3 activity (p < 0.05) but not cell death in RLMECs (p < 0.05). CONCLUSION: Staurosporine-induced disruption of the adherens junction and microvascular endothelial cell hyperpermeability is associated with the activation of mitochondrial "intrinsic" apoptotic signaling cascade but without causing endothelial cell death. Our results suggest that prevention of mitochondrial-mediated activation of caspase 3 has therapeutic potential against microvascular hyperpermeability.

Inhibition of Fas-Fas ligand interaction attenuates microvascular hyperpermeability following hemorrhagic shock.

Hemorrhagic shock (HS)-induced microvascular hyperpermeability poses a serious challenge in the management of trauma patients. Microvascular hyperpermeability occurs mainly because of the disruption of endothelial cell adherens junctions, where the "intrinsic" apoptotic signaling plays a regulatory role. The purpose of this study was to understand the role of the "extrinsic" apoptotic signaling molecules, particularly Fas-Fas ligand interaction in microvascular endothelial barrier integrity. Rat lung microvascular endothelial cells (RLMECs) were exposed to HS serum in the presence or absence of the Fas ligand inhibitor, FasFc. The effect of HS serum on Fas receptor and Fas ligand expression on RLMECs was determined by flow cytometry. Endothelial cell permeability was determined by monolayer permeability assay and the barrier integrity by ß-catenin immunofluorescence. Mitochondrial reactive oxygen species formation was determined using dihydrorhodamine 123 probe by fluorescent microscopy. Mitochondrial transmembrane potential was studied by fluorescent microscopy as well as flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Rat lung microvascular endothelial cells exposed to HS serum showed increase in Fas receptor and Fas ligand expression levels. FasFc treatment showed protection against HS serum-induced disruption of the adherens junctions and monolayer hyperpermeability (P < 0.05) in the endothelial cells. Pretreatment with FasFc also decreased HS serum-induced increase in mitochondrial reactive oxygen species formation, restored HS serum-induced drop in mitochondrial transmembrane potential, and reduced HS serum-induced caspase 3 activity in RLMECs. These findings open new avenues for drug development to manage HS-induced microvascular hyperpermeability by targeting the Fas-Fas ligand-mediated pathway.

ß-Catenin dynamics in the regulation of microvascular endothelial cell hyperpermeability.

ß-Catenin, a key regulator of barrier integrity, is an important component of the adherens junctional complex. Although the roles of ß-catenin in maintaining the adherens junctions and Wnt signaling are known, the dynamics of ß-catenin following insult and its potential role in vascular recovery/repair remain unclear. Our objective was to define ß-catenin's dynamics following disruption of the adherens junctional complex and subsequent recovery. Rat lung microvascular endothelial cells were treated with active caspase 3 enzyme, by protein transference method, as an inducer of junctional damage and permeability. The disruption and subsequent recovery of ß-catenin to the adherens junctions were studied via immunofluorescence. Rat lung microvascular endothelial cell monolayers were used to measure hyperpermeability. To understand the role of ß-catenin on nuclear translocation/transcriptional regulation in relationship to the recovery of the adherens junctions, Tcf-mediated transcriptional activity was determined. Active caspase 3 induced a loss of ß-catenin at the adherens junctions at 1 and 2 h followed by its recovery at 3 h. Transference of Bak peptide, an inducer of endogenous caspase 3 activation, induced hyperpermeability at 1 h followed by a significant decrease at 2 h. Inhibition of GSK-3ß and the transfection of ß-catenin vector increased Tcf-mediated transcription significantly (P < 0.05). The dissociated adherens junctional protein ß-catenin translocates into the cytoplasm, resulting in microvascular hyperpermeability followed by a time-dependent recovery and relocation to the cell membrane. Our data suggest a recycling pathway for ß-catenin to the cell junction.