Human Microvessel Endothelial Cells Research Articles

Abstract PO3-24-07: The role of Hepatitis A Virus Cellular Receptor 1 (HAVCR1) in breast cancer and the impact on penetration of blood brain barrier

Abstract Background: Hepatitis A Virus Cellular Receptor 1 (HAVCR1) is associated with the biological behaviour of several cancers, and has been shown to regulate junctional complexes. However, its role in breast cancer remains unexplored. This study aimed to investigate the effects of HAVCR1 on human breast cancer. Methods: We explored the UALCAN web portal, which is linked to the TCGA database, to determine the expression of HAVCR1 in breast cancer and the Kaplan-Meier plotter to evaluate the correlation with the survival of the patients. HAVCR1 siRNA was used to knock down the HAVCR1 expression in MCF-7 and MDA-MB-231 cell lines. In vitro cell growth, wounding healing, and electric cell impedance sensing (ECIS for assessing cellular proliferation, migration and adhesion) assays were used. Transepithelial resistance (TEER) and paracellular permeability (PCP) were assessed to determine changes in the barrier function of human breast cancer cells. The expression of HAVCR1 and prospective interactive binding molecules of HAVCR1 protein was assessed using quantitative polymerase chain reaction (qPCR). Cellular response to docetaxel was evaluated and shown (IC50). The invasiveness of breast cancer cells through the human cerebral microvessel endothelial cells (hCMEC/D3) cell layers was measured using an in vitro transwell cell invasion assay. Results: HAVCR1 gene expression levels were significantly lower (P< 0.001) in breast cancer tissue compared with normal tissues. Patients with high expression of HAVCR1 had a significantly better relapse-free survival than low expression (P< 0.001). There was no significant difference in overall survival (OS) between the HAVCR1 high and the low expression groups (P=0.22). However, in subgroup analysis, high HAVCR1 expression was associated with better OS in luminal A (P=0.04). Knockdown (KD) HAVCR1 in breast cancer cells influence cell function by enhanced adhesion, proliferation, and migration compared to wild type (WT) cell lines. It was noted that loss of HAVCR1 rendered both MCF-7 and MDA-MB-231 cells resistant to docetaxel (IC50 for WT and HAVCR1 KD respectively being 0.86nM vs 1.75nM for MCF-7 and 1.35nM vs 15.48nM for MDA-MB-231). HAVCR1 KD increase adhesion to hCMEC/D3 cells (53.14±8.23 vs ±99.37±8.97 for MCF-7, P< 0.001; 109.5±97.56 vs 147.1±96.63 for MDA-MB-231, P=0.228, WT and HAVCR1 KD). In the cerebral endothelial cell invasion assay, we found that HAVCR1 KD resulted in a significantly increased invasion of the breast cancer cells through the endothelium (19.27±8.74 vs 33.58±16.81 for MDA-MB-231, P=0.0114, WT and HAVCR1 KD). For the barrier function, the knockdown led to a decrease in TEER and an increase in PCP when compared with WT cells in both cell lines. HAVCR1 KD cells showed decreased barrier function, together with reduced expression of Claudin-1/8/10, occludin, MarvelD3, Tricellulin and ZO1. Conclusions: HAVCR1 is a potential biomarker for breast cancer prognosis. HAVCR1 influences the integrity of barrier function by influence the gene expression of tight junction in breast cancer, and influence the penetration to blood brain barrier. Together, it suggests that HAVCR1 may be a potential therapeutic target in breast cancer. Citation Format: Xiaoshan Cao, Binbin Cong, Wen Jiang, Tracey Martin. The role of Hepatitis A Virus Cellular Receptor 1 (HAVCR1) in breast cancer and the impact on penetration of blood brain barrier [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-07.

Dihydroartemisinin protects blood-brain barrier permeability during sepsis by inhibiting the transcription factor SNAI1.

Blood–brain barrier (BBB) injury is involved in the pathogenesis of sepsis‐associated encephalopathy. In this study, we used dihydroartemisinin (DHA), a derivative of artemisinin, to treat a cecal ligation and puncture (CLP)‐induced mouse sepsis model and a tumour necrosis factor α (TNF‐α)‐stimulated human cerebral microvessel endothelial cells (hCMEC)/D3 cell line. We found that DHA decreased BBB permeability and increased the expression of the tight junction protein occludin (OCLN) in the CLP model. In hCMEC/D3 cells, DHA decreased TNF‐α‐induced hyperpermeability and increased the expression of OCLN. DHA also repressed SNAI1 expression in the CLP mouse model and in TNF‐α‐stimulated hCMEC/D3 cells. These data suggest that DHA protects BBB permeability during sepsis by stimulating the expression of OCLN, by downregulating the expression of the SNAI1 transcription factor.

Sunitinib induced resistance of endothelial cells by up-regulating P-glycoprotein and PI3K/Akt pathway

Drug resistance is a crucial obstacle to achieve satisfactory chemotherapeutic effects. Numerous studies have shown that the PI3K/Akt signaling pathway plays a significant role in various processes of cellular events and tumor progression, while few studies have focused on the PI3K/Akt signaling pathway in drug resistance of endothelial cells. The present study aims to explore the relationship of PI3K/Akt signaling and cellular resistance to anticancer drugs in human microvessel endothelial cells (HMEC-1). We established stable sunitinib-resiatant human microvessel endothelial cells (HMEC-su) after long-term exposure to sunitinib (a small-molecule tyrosine kinase receptor inhibitor) for 12 months. HMEC-su showed significant alternations of cell morphology and exhibited a 2.32-fold higher IC50 of sunitinib than parental HMEC-1 cells. Expression of P-glycoprotein (P-gp) and breast cancer-resistance protein (ABCG2) which mediates drug efflux, increased significantly in HMEC-su lines compared with HMEC-1 cells by western blots assay. Our study further demonstrates that LY294002 (blocking the PI3K/Akt pathway) enhances the sensibility of HMEC-su to suntinib and inhibits the gene transcription and protein expression of P-gp, ABCG2 in HMEC-su cells. In conclusion, these results indicate that LY294002 could reverse P-gp and ABCG2 mediated-drug resistance to sunitinib in HMEC-su cells by inhibiting PI3K/Akt signaling.

POS0398 ADIPONECTIN INDUCES SYNOVIAL ANGIOGENESIS IN RHEUMATOID ARTHRITIS THROUGH METABOLIC REMODELING

Background:Our team have previously reported that Adiponectin correlates well with synovial inflammation and progressive bone erosion in rheumatoid arthritis (RA). Angiogenesis is another important part, which plays a critical role in the pathogenesis of RA.Objectives:We hypothesized that adiponectin induces synovial angiogenesis in RA.Methods:Single-cell RNA sequencing (scRNA-Seq) was used to screen cellular changes in local knee joint of collagen-induced arthritis (CIA) after intraarticularly injected of adiponectin. Chimera models of synovium-cartilage-NOD/SCID mice, matrigel plug assay and rat aortic ring assay were performed to demonstrate the pro-angiogenesis role of adiponectin. Cellular experiment, including proliferation, migration, apoptosis, tube formation and angiogenesis related gene expression profile, were detected with Human Umbilical Vein Endothelial Cells (HUVEC) and Mice Lung Microvessel Endothelial Cell (MLMEC) after adiponectin stimulation. Seahorse was performed to clear the influence of adiponectin to cell metabolism.Results:The synovium and pannus hyperplasia worse in CIA model after intraarticularly injected of adiponectin, along with more serious synovitis and bone erosion. ScRNA-Seq of synovial tissues separated from CIA reminded that endothelial cell barbarically grows via metabolic remodeling after stimulated with adiponectin. Synovial chimera, matrigel plug and rat aortic ring shows adiponectin accelerates angiogenesis significantly in different background conditions. In vitro, endothelial cell proliferation detecting by RCTA and CCK8, migration by wound healing and transwell, apoptosis by FACS, tube formation and angiogenesis related gene expression profile by PCR-ARRAY were promoted by adiponectin in both HUVEC and MLMEC. Seahorse showed HUVEC made more use of glycolysis after co-cultured with adiponectin, a method of cell energy supply that tumor cells possess called warburg effect, that drives endothelial cell hyperplasia in severe environment.Conclusion:As a classic metabolic regulator, adiponectin exacerbates CIA by promoting angiogenesis through metabolic remodeling. The findings not only provide a novel insight into the pathogenic role of adiponectin, but also reveals a potential therapeutical strategy to attenuate revascularization in RA.Disclosure of Interests:None declared

Molecular and Functional Study of Transient Receptor Potential Vanilloid 1-4 at the Rat and Human Blood-Brain Barrier Reveals Interspecies Differences.

Transient receptor potential vanilloid 1-4 (TRPV1-4) expression and functionality were investigated in brain microvessel endothelial cells (BMEC) forming the blood–brain barrier (BBB) from rat and human origins. In rat, Trpv1-4 were detected by qRT-PCR in the brain cortex, brain microvessels, and in primary cultures of brain microvessel endothelial cells [rat brain microvessel endothelial cells (rPBMEC)]. A similar Trpv1-4 expression profile in isolated brain microvessels and rPBMEC was found with the following order: Trpv4 > Trpv2 > Trpv3 > Trpv1. In human, TRPV1-4 were detected in the BBB cell line human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) and in primary cultures of BMEC isolated from human adult and children brain resections [human brain microvascular endothelial cells (hPBMEC)], showing a similar TRPV1-4 expression profile in both hCMEC/D3 cells and hPBMECs as follow: TRPV2 > > TRPV4 > TRPV1 > TRPV3. Western blotting and immunofluorescence experiments confirmed that TRPV2 and TRPV4 are the most expressed TRPV isoforms in hCMEC/D3 cells with a clear staining at the plasma membrane. A fluorescent dye Fluo-4 AM ester was applied to record intracellular Ca2+ levels. TRPV4 functional activity was demonstrated in mediating Ca2+ influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC50 of 16.2 ± 4.5 nM), which was inhibited by the specific TRPV4 antagonist, RN1734 (30 μM). In contrast, TRPV1 was slightly activated in hCMEC/D3 cells as shown by the weak Ca2+ influx induced by capsaicin at a high concentration (3 μM), a highly potent and specific TRPV1 agonist. Heat-induced Ca2+ influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 μM), in agreement with the low expression of TRPV1 as assessed by qRT-PCR. Our present study reveals an interspecies difference between Rat and Human. Functional contributions of TRPV1-4 subtype expression were not identical in rat and human tissues reflective of BBB integrity. TRPV2 was predominant in the human whereas TRPV4 had a larger role in the rat. This interspecies difference from a gene expression point of view should be taken into consideration when modulators of TRPV2 or TRPV4 are investigated in rat models of brain disorders.

Macrophage inhibitory cytokine-1 promotes angiogenesis by eliciting the GFRAL-mediated endothelial cell signaling.

Macrophage inhibitory cytokine-1 (MIC-1) is a cytokine with pleotropic actions and its expression is markedly increased by inflammation and cardiac injury and in cancers. In particular, MIC-1 production after cardiac ischemia injury is associated with enhanced cardiac angiogenesis as well as myocardial protection. However, it remains uncertain whether MIC-1 itself has proangiogenic activity. In this study, we tried to determine the precise role of MIC-1 in physiological and pathological angiogenesis. Human microvessel endothelial cells responded to MIC-1 with enhanced angiogenic behaviors. Employing various angiogenesis assays, MIC-1 was found to promote vessel formation and development with a potency similar to that of vascular endothelial growth factor (VEGF). MIC-1 transgenic (Tg) mice also displayed enhanced neovascularization in both developing embryos and neonatal mouse retinas, compared with wild-type mice. Furthermore, endothelial cells (ECs) isolated from MIC-1 Tg mouse lung exhibited higher angiogenic potential than ECs from wild-type lung. MIC-1-induced angiogenesis was also observed in the recovery or healing processes of injuries such as hindlimb ischemia and skin wounds in mice. However, unlike VEGF, MIC-1 induced neither endothelial inflammation nor increased vascular permeability. In ECs, the MIC-1 signal exerted proangiogenic actions via the MEK/extracellular signal-regulated kinase- and phosphatidylinositol 3-kinase/Akt-dependent pathways. Notably, these MIC-1 signaling events in ECs were abrogated by small interfering RNA-mediated knockdown of GFRAL, suggesting that GFRAL is an EC receptor for MIC-1. In summary, we here show a novel role of MIC-1 as a potent EC activator, which promotes both normal and injury-related angiogenesis.

Ultrasound in the presence of microbubbles in a dynamic invitro microfluidic blood-brain barrier model

Controllable, localized permeability of the blood-brain barrier (BBB) has been a goal of health professionals for enhanced delivery of drugs and therapeutic compounds for cancer and other neurological medical conditions. The transient permeability induced by transcranial ultrasound exposure in the presence of microbubbles has been shown to be a promising option, but little is known about the exact mechanism and response on a cellular level. A microfluidic device developed to recreate the in vivo microenvironment, including physiological fluid flow, was seeded with human cerebral microvessel endothelial cells surrounded by a matrix of astrocytes. The devices were exposed to ultrasound with and without the presence of microbubbles. The ultrasound parameters ranged in frequency from 0.5 to 1.55 MHz, duty factor from 0.0001–0.001 and pulse repetition frequency of 1 Hz–10 Hz. Microbubble size, composition, and concentration within the pumped fluid were also varied, along with varied flow rate. The transepithelial electrical resistance (TEER) and cell morphology were monitored throughout and post ultrasound exposure to understand the effects on permeability and cell viability. With this controlled variation of the ultrasound and microbubble parameters, a greater understanding of the induced permeability could lead to more success for clinical utilization.

Investigation of ultrasound exposure in the presence of microbubbles on a LumeNEXT blood-brain barrier model

The blood-brain barrier (BBB) is notoriously difficult to penetrate for the delivery of drugs and therapeutic compounds due to the unique nature of the tight junctions between endothelial cells. Ultrasound in the presence of microbubbles has been discovered to transiently open the BBB, however, little is known about the mechanism and the effects of excitation parameters at a cellular level. In this study, a static vascular lumen in vitro model, LumeNEXT, was utilized to investigate the excitation parameters in a controlled manner. Fibrin lumen hydrogels were lined with human cerebral microvessel endothelial and astrocyte cells. Cells were exposed to ultrasound with and without the presence of microbubbles. The devices were exposed to ultrasound parameters ranging in frequency from 0.5 to 1.55MHz, duty factor from 0.0001–0.001 and pulse repetition frequency of 1–10 Hz. Acoustic and thermal characterization of the device was performed. Effects of microbubble size and composition on the endothelial permeability were also investigated. The transepithelial electrical resistance (TEER) and cell morphology were monitored post-ultrasound exposure to investigate endothelial permeability duration and extent. Understanding the fundamentals of ultrasound-induced permeability in a controlled manner can lead to further investigations and optimization for in vivo and clinical settings.

Z-guggulsterone regulates MDR1 expression mainly through the pregnane X receptor-dependent manner in human brain microvessel endothelial cells

Recently studies showed that pregnane X receptor (PXR) was expressed in human brain microvessel endothelial cells and coordinately induced multidrug resistance protein 1 (MDR1) expression. The present study aimed to investigate the regulatory effect of Z-guggulsterone on MDR1 in human brain microvessel endothelial cells, and explored whether it involved modulation of PXR. The results showed that Z-guggulsterone (30 μM) simultaneously inhibited the expression of PXR and MDR1 at 24 h in human brain-derived microvessel endothelial cells (hBDMECs). Meanwhile, the levels of PXR and MDR1 expression were simultaneously reduced in PXR siRNA-transfected hBDMECs; MDR-1 siRNA-transfected hBDMECs showed significant decrease in MDR1 expression, but no change in PXR expression. Furthermore, Z-guggulsterone inhibited the activation of PXR in hBDMECs through decreasing the release of cAMP/PKA. Z-guggulsterone reduced the co-activator SRC-1 expression in hBDMECs, as to prevent the activation of MDR1 gene transcription. In addition, Z-guggulsterone (30 μM) at 24 h significantly inhibited the expression of human constitutive androstane receptor (CAR) protein in hBDMECs. However, after treatment with Z-guggulsterone (≤30 μM), the level of MDR1 reporter gene activity was lower in human PXR-transfected cells than that in human CAR-transfected cells. The inhibition effect of Z-guggulsterones on MDR1 reporter gene activation was gradually enhanced with the increase of human PXR to CAR ratio, which was greater extent than that with the increase of human CAR to hPXR ratio. The present study suggested that Z-guggulsterone down-regulating the efflux function and expression of MDR1 in hBDMECs might be mainly through the PXR-dependent manner.

Targeted Proteomics-Based Quantitative Protein Atlas of Pannexin and Connexin Subtypes in Mouse and Human Tissues and Cancer Cell Lines

The pannexin (Px) and connexin (Cx) families form multimeric hemichannels that mediate cellular transport of a wide variety of signaling and other molecules and exhibit pathophysiological and pharmacological functions. Twenty-four Px and Cx subtypes have been identified in humans and 23 in mice. The purpose of this study is to establish a quantitative protein atlas of Px and Cx subtypes in mouse and human tissues and cancer cell lines by means of quantitative targeted absolute proteomics, using an internal standard protein in which stable-isotope-labeled target peptides selected according to in silico criteria are concatenated together with internal reference peptides for the determination of the protein amount. This quantification system enabled us to cover 20 of 24 subtypes (83%) in humans, and 21 of 23 subtypes (91%) in mice. In mice, Px1, Cx32, and Cx43 were most abundantly expressed in the small intestine, liver and pancreas, and brain capillary, brain, and heart, respectively. Human blood-brain barrier endothelial cells (human cerebral microvessel endothelial cells) highly expressed Px1 and Cx43. Among human cancer cells, Panc-1 selectively expressed Px1, and Caco-2 cells abundantly expressed Cx32, while MCF-7 and AsPC-1 did not express any subtypes of hemichannels tested. These results suggest that Px1, Cx32, and Cx43 appear to play predominant roles in normal tissues and some cancer cells.

Amyloid beta25-35 impairs docosahexaenoic acid efflux by down-regulating fatty acid transport protein 1 (FATP1/SLC27A1) protein expression in human brain capillary endothelial cells.

Decreased levels of docosahexaenoic acid (DHA), an endogenous neuroprotective compound, in the brain are associated with the development of Alzheimer's disease (AD). We previously showed that DHA is a substrate of fatty acid transport protein 1 (FATP1/SLC27A1), and FATP1 is localized at the abluminal membrane of brain capillary endothelial cells. We hypothesized that amyloid β (Aβ) decreases FATP1-mediated cellular efflux (i.e. supply to the brain) of DHA at the blood-brain barrier (BBB). Here, we tested this hypothesis using a human cerebral microvascular endothelial cell line, human cerebral microvessel endothelial cells (hCMEC/D3), as a BBB model. The efflux of DHA-d5 by hCMEC/D3 cells increased time-dependently up to 3min. Knock-down of FATP1 with specific siRNA indicated that FATP1-mediated efflux accounts for 47.0% of this DHA-d5 efflux. In hCMEC/D3 cells treated with Aβ25-35 (10μM/24h), which we employed as an in vitro model of the BBB in AD, FATP1 protein expression in the plasma membrane was decreased by 96.0%, which was greater than the decrease in the whole-cell lysate, and the DHA-d5 efflux was decreased by 68.3%. Of this 68.3% decrease, 45.1% (47.0×0.96) is accounted for by the decrease in FATP1-mediated efflux and the remaining 23.2% is presumably mediated by other mechanism(s). Thus, we have established for the first time that FATP1 is a major contributor to DHA efflux from human brain capillary endothelial cells, and its efflux activity at the abluminal membrane of the cells is blocked by Aβ. This may explain the decreased DHA level in the brain of AD patients. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Disruption of endothelial barrier function is linked with hyposecretion and lymphocytic infiltration in salivary glands of Sjögren's syndrome

Sjögren's syndrome (SS) is an inflammatory autoimmune disease that causes hyposecretion in salivary glands. Endothelial tight junctions (TJs) play crucial roles in salivation and barrier function of blood vessels. However, whether the alteration of endothelial TJs were involved in pathogenesis of SS was still unknown. Here, the ultrastructure and function of endothelial TJs in submandibular glands (SMGs) were detected by transmission electron microscopy and in vivo paracellular permeability assay in different aged NOD mouse model for SS. CFSE-labeled lymphocytes were injected into tail vein to trace the infiltration, while claudin-5 expression and distribution were detected by immunofluorescence, qRT-PCR, and western blot. Results showed that the stimulated salivary flow rate was gradually decreased and lymphocytic infiltration was found as age increased in 12- and 21-week-old NOD mice, but not 7-week-old NOD mice. Blood vessels were dilated, while endothelial TJ width and paracellular tracer transport were increased in 12-week-old NOD mice. Moreover, the injected CFSE-labeled lymphocytes were observed in SMGs of 12-week-old NOD mice. Claudin-5 level was increased and relocalized from the apical portion of neighboring endothelial cells to lateral membranes and cytoplasm in 12-week-old NOD mice. Additionally, the alteration of claudin-5 expression and distribution was further confirmed in labial salivary glands and bilateral parotid glands from SS patients. In cultured human microvessel endothelial cell line (HMEC-1), IFN-γ stimulation significantly increased claudin-5 expression. Taken together, we identified that the endothelial TJ barrier was disrupted and contributed to the development of salivary hyposecretion and lymphocytic infiltration in SS.

Evaluation of brain-targeted chitosan nanoparticles through blood–brain barrier cerebral microvessel endothelial cells

The blood–brain barrier (BBB) is the major problem for the treatment of central nervous system diseases. A previous study from our group showed that the brain-targeted chitosan nanoparticles-loaded with large peptide moieties can rapidly cross the barrier and provide neuroprotection. The present study aims to determine the efficacy of the brain-targeted chitosan nanoparticles’ uptake by the human BBB cerebral microvessel endothelial cells (hCMECs) and to investigate the underlying mechanisms for enhanced cellular entry. Fluorescently labelled nanoparticles either conjugated with antibodies recognising human transferrin receptor (anti-TfR mAb) or not were prepared, characterised and their interaction with cerebral endothelial cells was evaluated. The antibody decoration of chitosan nanoparticles significantly increased their entry into hCMEC/D3 cell line. Inhibition of cellular uptake by chlorpromazine indicated that the anti-TfR mAb-conjugated nanoparticles were preferentially cell internalised through receptor-mediated endocytosis pathway. Alternatively, as primarily observed with control chitosan nanoparticles, aggregation of nanoparticles may also have induced macropinocytosis.

SSeCKS/AKAP12 induces repulsion between human prostate cancer and microvessel endothelial cells through the activation of Semaphorin 3F

Metastasis remains the primary cause of prostate cancer related death. Cancer cells need to contact endothelial cells and disrupt endothelial junctions to cross the endothelium for invasion and metastasis. The suppression of heterotypic repulsion between cancer and endothelial cells allows cancer cells to invade into the surrounding tissue. Here, we demonstrate that SSeCKS/AKAP12 induced repulsion between human prostate cancer and microvessel endothelial cells, which was mediated by an angiogenesis inhibitor Semaphorin 3F. Moreover, we examined AKAP12 and Semaphorin 3F mRNA expression in 42 prostate cancer and 30 benign prostatic hyperplasia tissue samples, and found that the expression of AKAP12 and Semaphorin 3F mRNA was inversely associated with the degree of aggressiveness of prostate cancer cells and tissues. An ordinal logistic regression analysis indicates that there is a positive association between the expression of AKAP12 and Semaphorin 3F in prostate cancer, suggesting that the activation of Semaphorin 3F by SSeCKS/AKAP12 may be involved in prostate cancer progression and metastasis.

Human Turbinate-derived Mesenchymal Stem Cells Differentiated into Keratocyte Progenitor Cells

Objective: Though keratoplasty is used to treat corneal blindness, donor shortage, tendency of stimulated keratocyte transformed to fibroblast and immunological rejection are still big problems. As a solution, cornea tissue engineering using non-corneal tissue sourced cells become emerging issue. Thus, this study was designed to find novel material for keratoplasty. Methods: Human turbinate-derived mesenchymal stem cells (hTMSCs) were obtained from patients and cultured with differentiation medium for 14 days. The keratocyte markers, stem cell markers, early corneal stromal stem cell (CSSC) markers, were measured by real time-PCR. The MSC markers were detected by FACS. Results: After 14 days of differentiation medium exposure, hTMSCs expressed markers of keratocyte such as keratocan sulfate proteoglycan (KERA) and aldehydrogenase (ALDH). As the hTMSCs became keratocytes, the expression of embryonic ocular precursor markers ABCG2 and PAX6 decreased but were still measurable. Early CSSC markers including SIX2, SIX3, BMI expression was elevated after 7 d and reduced after 14 d of KDM treatment. The stem cell markers such as SOX2, Notch were decreased. After 14 d of differentiation, the hTMSCs expressed MSC markers CD73, CD90, and CD105, but not hematopoietic markers CD14, CD19, CD34, HLA-DR; these changes indicate development of a characteristic MSC phenotype. hTMSCs inhibited the tube-formation ability of human microvessel endothelial cells. hTMSCs derived from neural crest could differentiate into keratocyte progenitors. Conclusions: This study first reveals that the hTMSCs have potential to be differentiated into keratocyte progenitor- like cells. Use of hTMSCs derived from neural crest in cell-based therapeutics as source for corneal tissue engineering may overcome the problems of keratoplasty such as immunological rejection and limiting supply of human donor corneas.

Development of a blood-brain barrier model in a membrane-based microchip for characterization of drug permeability and cytotoxicity for drug screening

Since most of the central nervous system (CNS) drug candidates show poor permeability across the blood-brain barrier (BBB), development of a reliable platform for permeability assay will greatly accelerate drug discovery. Herein, we constructed a microfluidic BBB model to mimic drug delivery into the brain to induce cytotoxicity at target cells. To reconstitute the in vivo BBB properties, human cerebral microvessel endothelial cells (hCMEC/D3) were dynamically cultured in a membrane-based microchannel. Sunitinib, a model drug, was then delivered into the microchannel and forced to permeate through the BBB model. The permeated amount was directly quantified by an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS) after on-chip SPE (μSPE) pretreatment. Moreover, the permeated drug was incubated with glioma cells (U251) cultured inside agarose gel in the downstream to investigate drug-induced cytotoxicity. The resultant permeability of sunitinib was highly correlated with literature reported value, and it only required 30 min and 5 μL of sample solution for each permeation experiment. Moreover, after 48 h of treatment, the survival rate of U251 cells cultured in 3D scaffolds was nearly 6% higher than that in 2D, which was in accordance with the previously reported results. These results demonstrate that this platform provides a valid tool for drug permeability and cytotoxicity assays which have great value for the research and development of CNS drugs.

The effect of soluble E-selectin on tumor progression and metastasis.

BackgroundDistant metastasis resulting from vascular dissemination of cancer cells is the primary cause of mortality from breast cancer. We have previously reported that E-selectin expression on the endothelial cell surface mediates shear-resistant adhesion and migration of circulating cancer cells via interaction with CD44. As a result of shedding, soluble E-selectin (sE-selectin) from the activated endothelium is present in the serum. In this study, we aimed to understand the role of sE-selectin in tumor progression and metastasis.MethodsWe investigated the effect of sE-selectin on shear-resistant adhesion and migration of metastatic breast cancer cells and leukocytes in vitro and in vivo.ResultsWe found that sE-selectin promoted migration and shear-resistant adhesion of CD44+/high breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to non-activated human microvessel endothelial cells (ES-HMVECs), but not of CD44-/low breast cancer cell lines (MCF-7 and T-47D). This endothelial E-selectin independent, sE-selectin-mediated shear-resistant adhesion was also observed in a leukocyte cell line (HL-60) as well as human peripheral blood mononuclear cells (PBMCs). Additionally, the incubation of MDA-MB-231 cells with sE-selectin triggered FAK phosphorylation and shear-resistant adhesion of sE-selectin-treated cells resulted in increased endothelial permeabilization. However, CD44 knockdown in MDA-MB-231 and HL-60 cells resulted in a significant reduction of sE-selectin-mediated shear-resistant adhesion to non-activated HMVECs, suggesting the involvement of CD44/FAK. Moreover, functional blockade of ICAM-1 in non-activated HMVECs resulted in a marked reduction of sE-selectin-mediated shear-resistant adhesion. Finally, the pre-incubation of CD44+ 4 T1 murine breast cancer cells with sE-selectin augmented infiltration into the lung in E-selectin K/O mice and infusion of human PBMCs pre-incubated with sE-selectin stimulated MDA-MB-231 xenografted breast tumor growth in NSG mice.ConclusionsOur data suggest that circulating sE-selectin stimulates a broad range of circulating cells via CD44 and mediates pleiotropic effects that promote migration and shear-resistant adhesion in an endothelial E-selectin independent fashion, in turn accelerating tissue infiltration of leukocytes and cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2366-2) contains supplementary material, which is available to authorized users.

Unconjugated bilirubin elevation impairs the function and expression of breast cancer resistance protein (BCRP) at the blood-brain barrier in bile duct-ligated rats.

Liver failure is associated with dyshomeostasis of efflux transporters at the blood-brain barrier (BBB), which contributes to hepatic encephalopathy. In this study we examined whether breast cancer resistance protein (BCRP), a major efflux transporter at the BBB, was altered during liver failure in rats. Rats underwent bile duct ligation (BDL) surgery, and then were sacrificed after intravenous injection of prazosin on d3, d7 and d14. The brains and blood samples were collected. BCRP function at the BBB was assessed by the brain-to-plasma prazosin concentration ratio; Evans Blue extravasation in the brain tissues was used as an indicator of BBB integrity. The protein levels of BCRP in the brain tissues were detected. Human cerebral microvessel endothelial cells (HCMEC/D3) and Madin-Darby canine kidney cells expressing human BCRP (MDCK-BCRP) were tested in vitro. In addition, hyperbilirubinemia (HB) was induced in rats by intravenous injection of unconjugated bilirubin (UCB). BDL rats exhibited progressive decline of liver function and HB from d3 to d14. In the brain tissues of BDL rats, both the function and protein levels of BCRP were progressively decreased, whereas the BBB integrity was intact. Furthermore, BDL rat serum significantly decreased BCRP function and protein levels in HCMEC/D3 cells. Among the abnormally altered components in BDL rat serum tested, UCB (10, 25 μmol/L) dose-dependently inhibit BCRP function and protein levels in HCMEC/D3 cells, whereas 3 bile acids (CDCA, UDCA and DCA) had no effect. Similar results were obtained in MDCK-BCRP cells and in the brains of HB rats. Correlation analysis revealed that UCB levels were negatively correlated with BCRP expression in the brain tissues of BDL rats and HB rats as well as in two types of cells tested in vitro. UCB elevation in BDL rats impairs the function and expression of BCRP at the BBB, thus contributing to hepatic encephalopathy.

Radiation promotes malignant progression of glioma cells through HIF-1alpha stabilization

Given its contribution to malignant phenotypes of cancer, tumor hypoxia has been considered as a potential therapeutic problem. In the stressful microenvironment condition, hypoxia inducible factor 1 (HIF1) is well known to mediate the transcriptional adaptation of cells to hypoxia and acts as a central player for the process of hypoxia-driven malignant cancer progression. Here, we found that irradiation causes the HIF1α protein to stabilize, even in normoxia condition through activation of p38 MAPK, thereby promoting angiogenesis in tumor microenvironment and infiltrative property of glioma cells. Notably, irradiation reduced hydroxylation of HIF1α through destabilization of prolyl hydroxylases (PHD)-2. Moreover, radiation also decreased the half-life of protein von Hippel–Lindau (pVHL), which is a specific E3 ligase for HIF1α. Of note, inhibition of p38 MAPK attenuated radiation-induced stabilization of HIF1α through destabilization of PHD-2 and pVHL. In agreement with these results, targeting of either p38 MAPK, HIF1α, pVHL or PHD-2 effectively mitigated the radiation-induced tube formation of human brain-derived micro-vessel endothelial cells (HB-MEC) and infiltration of glioma cells. Taken together, our findings suggest that targeting HIF1α in combination with ionizing radiation might increase the efficacy of radiotherapy for glioma treatment.

Tumor endothelial expression of P-glycoprotein upon microvesicular transfer of TrpC5 derived from adriamycin-resistant breast cancer cells

Treatment of carcinoma commonly fails due to chemoresistance. Studies have shown that endothelial cells acquire resistance via the tumor microenvironment. Microvesicle (MV) shedding from the cell membrane to the microenvironment plays an important role in communication between cells. The aim of the present study was to determine whether MCF-7 adriamycin-resistant cells (MCF-7/ADM) shed MVs that alter the characteristics of human microvessel endothelial cells (HMECs). MVs from tumor cells transferred a Ca2+-permeable channel TrpC5 to HMECs, inducing the expression of P-glycoprotein (P-gp) by activation of the transcription factor NFATc3 (nuclear factor of activated T cells isoform c3). Expression of the mdr1 gene was blocked by the TrpC5-blocking antibody T5E3, and the production of P-gp in HMECs was reduced by blockade of TrpC5. Thus, we postulate that endothelial cells acquire the resistant protein upon exposure to TrpC5-containg MVs in the microenvironment, and express P-gp in the TrpC5–NFATc3 signal pathway.