scholarly journals Mitofusin-2 Triggers Cervical Carcinoma Cell Hela Apoptosis via Mitochondrial Pathway in Mouse Model

2018 ◽  
Vol 46 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Weiqiong Wang ◽  
Xiaowen Liu ◽  
Xiaomei Guo ◽  
Huanhuan Quan
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Western Blot ◽  
Cervical Carcinoma ◽  
Hela Cells ◽  
Mitochondrial Pathway ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Mitofusin 2

Background/Aims: Cervical carcinoma continues to be one of the most dangerous cancer types, and more effective therapies are urgently needed for cervical carcinoma treatment. Mitochondria-associated Mitofusin 2 has influence on the progression of many cancers. In the current study, we aimed to focus on the cell apoptotic effects of Mfn2 on cervical carcinoma HeLa cells in vitro and to try to explore its underlying mechanisms. Moreover, we investigated the anticancer potential of Mfn2 in a cervical carcinoma mouse model. Methods: Adenovirus-Mfn2 (Adv-Mfn2) was used to deliver mfn2 into HeLa cells and tumour tissues in a nude mouse model. CCK-8, TUNEL assay, Western blot and immunohistochemical staining were performed to detect the effects of Mfn2. The mRNA level of Mfn2 was determined by quantitative realtime PCR (qRT-PCR) analysis. The effect of Mfn2 on cell apoptosis was investigated by flow cytometry. Flow cytometry was used to assess the change of the mitochondrial membrane potential of the cells treated with JC-1 assay. Mfn2, Bax, Bcl-2, cytochrome c, cleaved caspase-3, and cleaved caspase-9 protein levels were analysed by Western blot. Results: Data from CCK-8 and flow cytometry showed that Mfn2 could inhibit proliferation and induce apoptosis in a dose- and time-dependent manner in HeLa cells. JC-1 test results revealed that the membrane potential of the mitochondrial decreased in a dose-dependent manner in HeLa cells after Adv-Mfn2 treatment. The data from Western blot confirmed that higher cytosolic amounts of cytochrome c with increasing doses of Adv-Mfn2 signified the onset of the intrinsic apoptotic pathway. Levels of cleaved caspase-3 and cleaved caspase-9 increased in HeLa cells with Adv-Mfn2 treatment. We also found significant increases in the Bax level and a decreased Bcl-2 level with Adv-Mfn2 treatment. We further confirmed that Mfn2 could significantly inhibit the growth of the cervical tumour in the xenografted cervical carcinoma mouse model. After a 9-day-treatment, the tumours of the Adv-mfn2 group were inhibited and induced into apoptosis. The results demonstrated that the overexpression of Mfn2 could not only increase the levels of Bax and Bid in cervical tumour cells but also decrease the phosphorylation of Bad and the expression of Bcl-2. Conclusion: These studies suggested that the overexpression of Mfn2 could trigger cervical tumour apoptosis in vitro and in vivo, which was related to the mitochondrial pathway, and may provide a new treatment target for cervical carcinoma.

scholarly journals Rapid Apoptosis Induced by Shiga Toxin in HeLa Cells

2003 ◽  
Vol 71 (5) ◽  
pp. 2724-2735 ◽  
Author(s):  
Jun Fujii ◽  
Takashi Matsui ◽  
Daniel P. Heatherly ◽  
Kailo H. Schlegel ◽  
Peter I. Lobo ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Hela Cells ◽  
Shiga Toxin ◽  
Specific Inhibitor ◽  
Mitochondrial Pathway ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Mitochondrial Membranes ◽  
Caspase 9 ◽  
Induced Apoptosis

ABSTRACT Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.

scholarly journals Tanshinone IIA suppresses the progression of lung adenocarcinoma through regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ziheng Li ◽  
Ying Zhang ◽  
Yuan Zhou ◽  
Fuqian Wang ◽  
Chao Yin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Lung Adenocarcinoma ◽  
Annexin V ◽  
Tanshinone Iia ◽  
Dependent Manner ◽  
Information Analysis ◽  
Cdk2 Complex ◽  
Dual Staining

AbstractLung adenocarcinoma (LUAD) belongs to a subgroup of non-small cell lung cancer (NSCLC) with an increasing incidence all over the world. Tanshinone IIA (TSA), an active compound of Salvia miltiorrhiza Bunge., has been found to have anti-tumor effects on many tumors, but its anti-LUAD effect and its mechanism have not been reported yet. In this study, bio-information analysis was applied to characterize the potential mechanism of TSA on LUA, biological experiments were used to verify the mechanisms involved. TCGA, Pubchem, SwissTargetPrediction, Venny2.1.0, STRING, DAVID, Cytoscape 3.7.2, Omicshare, GEPIA, RSCBPDB, Chem Draw, AutoDockTools, and PyMOL were utilized for analysis in the bio-information analysis and network pharmacology. Our experiments in vitro focused on the anti-LUAD effects and mechanisms of TSA on LUAD cells (A549 and NCI-H1975 cells) via MTT, plate cloning, Annexin V-FITC and PI dual staining, flow cytometry, and western blot assays. A total of 64 differentially expressed genes (DEGs) of TSA for treatment of LUAD were screened out. Gene ontology and pathway analysis revealed characteristic of the DEGs network. After GEPIA-based DEGs confirmation, 46 genes were considered having significant differences. Further, 10 key DEGs (BTK, HSD11B1, ADAM33, TNNC1, THRA, CCNA2, AURKA, MIF, PLK1, and SORD) were identified as the most likely relevant genes from overall survival analysis. Molecular Docking results showed that CCNA2, CDK2 and PLK1 had the lowest docking energy. MTT and plate cloning assays results showed that TSA inhibited the proliferation of LUAD cells in a concentration-dependent manner. Annexin V-FITC and PI dual staining and flow cytometry assays results told that TSA promoted the apoptosis of the two LUAD cells in different degrees, and induced cycle arrest in the G1/S phase. Western blot results showed that TSA significantly down-regulated the expression of CCNA2, CDK2, AURKA, PLK1, and p-ERK. In summary, TSA could suppress the progression of LUAD by inducing cell apoptosis and arresting cell cycle, and these were done by regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway. These findings are the first to demonstrate the molecular mechanism of TSA in treatment of LUAD combination of network bio-information analysis and biological experiments in vitro.

Arsenic Trioxide Induces Platelet Apoptosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5149-5149
Author(s):  
Yicun Wu ◽  
Jin Dai ◽  
Weilin Zhang ◽  
Rong Yan ◽  
Changgeng Ruan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Arsenic Trioxide ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Pulmonary Haemorrhage ◽  
Tumour Cells ◽  
Dependent Manner ◽  
Platelet Apoptosis

Abstract Abstract 5149 Objective: Arsenic trioxide, a component of Traditional Chinese Medicine, is known as an effective anticancer drug especially in the treatment of acute promyelocytic leukaemia (APL). APL has emerged as the most curable subtype of acute myeloid leukaemia since the widely use of arsenic trioxide-based chemotherapy. However, recent researches show that thrombocytopenia occurred in 79% of the relapsed or refractory APL patients treated with arsenic trioxide, and part of the APL patients had to be stopped treatment because of catastrophic bleeding, such as intracranial and pulmonary haemorrhage. Thrombocytopenia also occurred in 43% of the myelodysplastic syndrome patients treated with arsenic trioxide. Recently, arsenic trioxide has been proved to have a pro-apoptotic effect on various kinds of nucleated tumour cells or non-tumour cells. The effect of arsenic trioxide on enucleated platelet, however, still remains unclear. The aim of current study is to investigate whether arsenic trioxide induces platelet apoptosis. Methods: Washed platelets (3 × 108/ml) were incubated with different concentrations of arsenic trioxide or vehicle at 37°C for 4 hours. Then, mitochondrial inner transmembrane potential (ΔΨm) and phosphatidylserine (PS) exposure were tested by flow cytometry. In the mean time, the treated platelets were analyzed by western blot for the expression levels of pro-apoptotic protein (Bax), and anti-apoptotic proteins (Bcl-2 and Bcl-XL). Activation of caspase-3 was also examined by western blot using an anti-caspase-3 antibody. Results: ΔΨm depolarization and PS exposure were dose-dependently induced in platelets incubated with different concentrations (2 uM, 4 uM, 8 uM, 16 uM) of arsenic trioxide as detected by flow cytometry, and the lowest concentration of arsenic trioxide incurring ΔΨm depolarization and PS exposure was 4 umol/L. Simultaneously, arsenic trioxide induced up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL in a dose-dependent manner. Furthermore, 17 kD cleaved caspase-3 fragments were dose-dependently induced in platelets treated with different concentrations of arsenic trioxide indicating that caspase-3 was activated by arsenic trioxide. Conclusions: Taken together, the data indicate that arsenic trioxide induces platelet apoptosis in vitro, which might suggest a novel pathogenic mechanism of thrombocytopenia in the patients who treated with arsenic trioxide. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel Imidazopyridine Derivative Exerts Anticancer Activity by Inducing Mitochondrial Pathway-Mediated Apoptosis

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Juanli Wang ◽  
Hong Wu ◽  
Guiting Song ◽  
Donglin Yang ◽  
Jiuhong Huang ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Hela Cells ◽  
Mitochondrial Pathway ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Cervical Cancer Cells ◽  
Related Proteins ◽  
Human Cervical Cancer

Background. Cancer remains a major clinical challenge because of the lack of effective drug for its treatment. To find out novel cancer chemotherapeutic molecules, we explored the anticancer effect of novel imidazopyridine compound 9i and also investigated the underlying molecular mechanism. Methods. Human cervical cancer cell (HeLa) viability was measured by an MTT assay after treatment with compound 9i. Clonogenicity of HeLa cells was investigated by an in vitro colony formation assay. Cell death was visualized by propidium iodide (PI) staining. Fluorescence-activated cell sorting (FACS) was used to determine apoptosis and mitochondrial membrane potential in HeLa cells. The expression level of apoptosis-related proteins was also determined by western blot. Results. Compound 9i suppressed HeLa cell viability in a time- and dose-dependent manner. Compound 9i induced mitochondrial outer membrane permeabilization (MOMP), activated caspase cascade, and finally resulted in apoptosis. Conclusion. Compound 9i induces mitochondrial pathway-mediated apoptosis in human cervical cancer cells, suggesting that 9i could be a potential lead compound to be developed as a cancer therapeutic molecule.

VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Aikebaier Maimaiti ◽  
Amier Aili ◽  
Hureshitanmu Kuerban ◽  
Xuejun Li
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Gallic Acid ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Dependent Manner ◽  
Lung Carcinoma Cells ◽  
Induced Apoptosis ◽  
The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

scholarly journals MODL-12. DEVELOPMENT OF A NOVEL IMMUNOCOMPETENT MOUSE MODEL FOR DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii413-iii413
Author(s):  
Maggie Seblani ◽  
Markella Zannikou ◽  
Katarzyna Pituch ◽  
Liliana Ilut ◽  
Oren Becher ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Growth Factor Receptor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Time Of Application ◽  
Tumor Associated Antigen ◽  
Sarcoma Virus

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor affecting young children. Immunotherapies hold promise however the lack of immunocompetent models recreating a faithful tumor microenvironment (TME) remains a challenge for development of targeted immunotherapeutics. We propose to generate an immunocompetent DIPG mouse model through induced overexpression of interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen overexpressed by glioma cells. A model with an intact TME permits comprehensive preclinical assessment of IL13Rα2-targeted immunotherapeutics. Our novel model uses the retroviral avian leucosis and sarcoma virus (RCAS) for in vivo gene delivery leading to IL13Rα2 expression in proliferating progenitor cells. Transfected cells expressing IL13Rα2 and PDGFB, a ligand for platelet derived growth factor receptor, alongside induced p53 loss via the Cre-Lox system are injected in the fourth ventricle in postnatal pups. We validated the expression of PDGFB and IL13Rα2 transgenes in vitro and in vivo and will characterize the TME through evaluation of the peripheral and tumor immunologic compartments using immunohistochemistry and flow cytometry. We confirmed expression of transgenes via flow cytometry and western blotting. Comparison of survival dynamics in mice inoculated with PDGFB alone with PDGFB+IL13Rα2 demonstrated that co-expression of IL13Rα2 did not significantly affect mice survival compared to the PDGFB model. At time of application, we initiated experiments to characterize the TME. Preliminary data demonstrate establishment of tumors within and adjacent to the brainstem and expression of target transgenes. Preclinical findings in a model recapitulating the TME may provide better insight into outcomes upon translation to clinical application.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

scholarly journals Ascorbic acid attenuates activation and cytokine production in sepsis-like monocytes

2021 ◽  
Author(s):  
Tobias Schmidt ◽  
Robin Kahn ◽  
Fredrik Kahn
Keyword(s):  
Flow Cytometry ◽  
Ascorbic Acid ◽  
Cytokine Production ◽  
In Vitro Study ◽  
Surface Expression ◽  
High Dose ◽  
Functional Assay ◽  
Dependent Manner ◽  
Dose Dependent

Objective To investigate the effects of high dose ascorbic acid (AA) on monocyte polarization and cytokine production in vitro Design Experimental in vitro study of cells from healthy subjects and patients with sepsis Setting University research laboratory and academic hospital Subjects Six healthy controls and three patients with sepsis Interventions Monocytes were isolated from whole blood of healthy donors (n=6) and polarized in vitro for 48hrs using LPS or LTA. Polarization was confirmed by surface marker expression using flow cytometry. As a comparison, monocytes were also isolated from septic patients (n=3) and analyzed for polarization markers. The effect of AA on monocyte polarization was evaluated. As a functional assay, AA-treated monocytes were analyzed for cytokine production of TNF and IL-8 by intracellular staining and flow cytometry following activation with LPS or LTA. Measurements and Main Results Both LPS and LTA induced polarization in healthy monocytes in vitro, with increased expression of both pro- (CD40 and PDL1, p<0.05) and anti-inflammatory (CD16 and CD163, p<0.05) polarization markers, with non-significant effects on CD86 and CD206. This pattern resembled, at least partly, that of monocytes from septic patients. Treatment with AA significantly inhibited the upregulation of surface expression of CD16 and CD163 (p<0.05) in a dose dependent manner, but not CD40 or PDL-1. Finally, AA attenuated LPS or LTA-induced cytokine production of IL-8 and TNF in a dose-dependent manner (both p<0.05). Conclusions AA inhibits upregulation of anti-, but not pro-inflammatory related markers in LPS or LTA polarized monocytes. Additionally, AA attenuates cytokine production from in vitro polarized monocytes, displaying functional involvement. This study provides important insight into the immunological effects of high dose AA on monocytes, and potential implications in sepsis.

scholarly journals Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246197
Author(s):  
Jorge Marquez ◽  
Jianping Dong ◽  
Chun Dong ◽  
Changsheng Tian ◽  
Ginette Serrero
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Negative Regulator ◽  
Target Validation ◽  
Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

Shear Stress Modulates the Expression of Thrombospondin-1 and CD36 in Endothelial Cells in vitro and during Shear Stress-Induced Angiogenesis in vivo

2006 ◽  
Vol 19 (1) ◽  
pp. 205873920601900 ◽  
Author(s):  
M. Bongrazio ◽  
L. DA Silva-Azevedo ◽  
E.C. Bergmann ◽  
O. Baum ◽  
B. Hinz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Western Blot ◽  
Dependent Manner ◽  
Vascular Networks ◽  
Thrombospondin 1 ◽  
Matrix Bound ◽  
Stress Dependent

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/1 drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress down-regulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

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