VhH anti-thrombomodulin clone 1 inhibits TAFI activation and enhances fibrinolysis in human whole blood under flow

Background: Thrombomodulin on endothelial cells can form a complex with thrombin. This complex has both anticoagulant properties, by activating protein C, and clot-protective properties, by activating thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI (TAFIa) inhibits plasmin-mediated fibrinolysis. Objectives: TAFIa inhibition is considered a potential antithrombotic strategy. So far, this goal has been pursued by developing compounds that directly inhibit TAFIa. In contrast, we here describe variable domain of heavy-chain-only antibody (VhH) clone 1 that inhibits TAFI activation by targeting human thrombomodulin. Methods: Two llamas (Lama Glama) were immunized, and phage display was used to select VhH anti-thrombomodulin (TM) clone 1. Affinity was determined with surface plasmon resonance and binding to native TM was confirmed with flow cytometry. Clone 1 was functionally assessed by competition, clot lysis, and thrombin generation assays. Last, the effect of clone 1 on tPA-mediated fibrinolysis in human whole blood was investigated in a microfluidic fibrinolysis model. Results: VhH anti-TM clone 1 bound recombinant TM with a binding affinity of 1.7 ± 0.4 nM and showed binding to native TM. Clone 1 competed with thrombin for binding to TM and attenuated TAFI activation in clot lysis assays and protein C activation in thrombin generation experiments. In a microfluidic fibrinolysis model, inhibition of TM with clone 1 fully prevented TAFI activation. Discussion: We have developed VhH anti-TM clone 1, which inhibits TAFI activation and enhances tPA-mediated fibrinolysis under flow. Different from agents that directly target TAFIa, our strategy should preserve direct TAFI activation via thrombin

Platelet function is disturbed by the angiogenesis inhibitors sunitinib and sorafenib, but unaffected by bevacizumab

Introduction: At the clinical introduction of antiangiogenic agents as anticancer agents, no major toxicities were expected as merely just endothelial cells (ECs) in tumors would be affected. However, several (serious) toxicities became apparent, of which underlying mechanisms are largely unknown. We investigated to what extent sunitinib (multitargeted antiangiogenic tyrosine kinase inhibitor (TKI)), sorafenib (TKI) and bevacizumab [specific antibody against vascular endothelial growth factor (VEGF)] may impair platelet function, which might explain treatment-related bleedings. Materials and methods: In vitro, the influence of sunitinib, sorafenib, and bevacizumab on platelet aggregation, P-selectin expression and fibrinogen binding, platelet–EC interaction, and tyrosine phosphorylation of c-Src was studied by optical aggregation, flow cytometry, real-time perfusion, and western blotting. Ex vivo, platelet aggregation was analyzed in 25 patients upon sunitinib or bevacizumab treatment. Concentrations of sunitinib, VEGF, and platelet and EC activation markers were measured by LC–MS/MS and ELISA. Results: In vitro, sunitinib and sorafenib significantly inhibited platelet aggregation (20 μM sunitinib: 71.3%, p < 0.001; 25 μM sorafenib: 55.8%, p = 0.042). Sorafenib and sunitinib significantly inhibited P-selectin expression on platelets. Exposure to both TKIs resulted in a reduced tyrosine phosphorylation of c-Src. Ex vivo, within 24 h sunitinib impaired platelet aggregation (83.0%, p = 0.001, N = 8). Plasma concentrations of sunitinib, VEGF, and platelet/EC activation markers were not correlated with disturbed aggregation. In contrast, bevacizumab only significantly impaired platelet aggregation in vitro at high c

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy

Background and aim: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. Methods: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. Results: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. Conclusions: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy

Background and aim: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. Methods: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. Results: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. Conclusions: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization

Variation at the paraoxonase gene locus contributes to carotid arterial wall thickness in subjects with familial hypercholesterolemia

Objectives: Paraoxonase (PON1) is a potent enzyme, physically associated with the high-density lipoprotein particle. PON1 may protect against cardiovascular disease (CVD), since it is capable of hydrolyzing oxidized LDL-cholesterol, thereby negating the detrimental effects of this lipoprotein on the arterial wall. Design and methods: In 187 patients with familial hypercholesterolemia, we studied the seven most common single nucleotide polymorphisms (SNPs) in both the coding and promoter sequences of PON1 (L55M, Q192R, T-107C, C-126G, G-162A, G-824A, and C-907G) in terms of PON1 activity and intima media thickness (IMT) of the carotid arterial wall, a validated surrogate marker for CVD. Results: In concordance with several previous studies, we observed that the L55M, T-107C, G-162A, G-824A, and C-907G SNPs conferred PON1 activity towards phenylacetate, while this was not the case for the Q192R and C-126G SNPs. Importantly, in a multivariate regression analysis, G-824A proved to be an independent predictor of carotid IMT. Additionally, the two fully discordant homozygous haplotypes, C-907/G-824/G-162/C-126/T-107/55M versus -907G/-824A/-162A/-126G/-107C/L55, differed by 22% in carotid IMT (P = 0.007). Conclusions: Genetic variation at the PON1 locus has a strong influence on PON1 activity as well as on carotid IMT. These data indicate that PON1 is indeed involved in the pathogenesis of atherosclerosis. Whether this also translates into a role for PON1 in the occurrence of CVD events needs to be confirmed by large prospective studies in the general population. (C) 2004 The Canadian Society of Clinical Chemists. All rights reserve

Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro

Extracellular vesicles (EV) secreted in the tumour microenvironment (TME) are emerging as major antagonists of anticancer therapies by orchestrating the therapeutic outcome through altering the behaviour of recipient cells. Recent evidence suggested that chemotherapeutic drugs could be responsible for the EV-mediated tumour-stroma crosstalk associated with cancer cell drug resistance. Here, we investigated the capacity of tumour EV (TEV) secreted by normoxic and hypoxic (1% oxygen) C26 cancer cells after doxorubicin (DOX) treatment to alter the response of naïve C26 cells and RAW 264.7 macrophages to DOX. We observed that C26 cells were less responsive to DOX treatment under normoxia compared to hypoxia, and a minimally cytotoxic DOX concentration that mounted distinct effects on cell viability was selected for TEV harvesting. Homotypic and heterotypic pretreatment of naïve hypoxic cancer and macrophage-like cells with normoxic DOX-elicited TEV rendered these cells slightly less responsive to DOX treatment. The observed effects were associated with strong hypoxia-inducible factor 1-alpha (HIF-1α) induction and B-cell lymphoma-extra-large anti-apoptotic protein (Bcl-xL)-mediated anti-apoptotic response in normoxic DOX-treated TEV donor cells, being also tightly connected to the DOX-TEV-mediated HIF-1α induction, as well as Bcl-xL levels increasing in recipient cells. Altogether, our results could open new perspectives for investigating the role of chemotherapy-elicited TEV in the colorectal cancer TME and their modulatory actions on promoting drug resistance

Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro

Extracellular vesicles (EV) secreted in the tumour microenvironment (TME) are emerging as major antagonists of anticancer therapies by orchestrating the therapeutic outcome through altering the behaviour of recipient cells. Recent evidence suggested that chemotherapeutic drugs could be responsible for the EV-mediated tumour-stroma crosstalk associated with cancer cell drug resistance. Here, we investigated the capacity of tumour EV (TEV) secreted by normoxic and hypoxic (1% oxygen) C26 cancer cells after doxorubicin (DOX) treatment to alter the response of naïve C26 cells and RAW 264.7 macrophages to DOX. We observed that C26 cells were less responsive to DOX treatment under normoxia compared to hypoxia, and a minimally cytotoxic DOX concentration that mounted distinct effects on cell viability was selected for TEV harvesting. Homotypic and heterotypic pretreatment of naïve hypoxic cancer and macrophage-like cells with normoxic DOX-elicited TEV rendered these cells slightly less responsive to DOX treatment. The observed effects were associated with strong hypoxia-inducible factor 1-alpha (HIF-1α) induction and B-cell lymphoma-extra-large anti-apoptotic protein (Bcl-xL)-mediated anti-apoptotic response in normoxic DOX-treated TEV donor cells, being also tightly connected to the DOX-TEV-mediated HIF-1α induction, as well as Bcl-xL levels increasing in recipient cells. Altogether, our results could open new perspectives for investigating the role of chemotherapy-elicited TEV in the colorectal cancer TME and their modulatory actions on promoting drug resistance

Endothelial Cell Targeting by cRGD-Functionalized Polymeric Nanoparticles under Static and Flow Conditions

Since &alpha;v&beta;3 integrin is a key component of angiogenesis in health and disease, Arg-Gly-Asp (RGD) peptide-functionalized nanocarriers have been investigated as vehicles for targeted delivery of drugs to the &alpha;v&beta;3 integrin-overexpressing neovasculature of tumors. In this work, PEGylated nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA) functionalized with cyclic-RGD (cRGD), were evaluated as nanocarriers for the targeting of angiogenic endothelium. For this purpose, NPs (~300 nm) functionalized with cRGD with different surface densities were prepared by maleimide-thiol chemistry and their interactions with human umbilical vein endothelial cells (HUVECs) were evaluated under different conditions using flow cytometry and microscopy. The cell association of cRGD-NPs under static conditions was time-, concentration- and cRGD density-dependent. The interactions between HUVECs and cRGD-NPs dispersed in cell culture medium under flow conditions were also time- and cRGD density-dependent. When washed red blood cells (RBCs) were added to the medium, a 3 to 8-fold increase in NPs association to HUVECs was observed. Moreover, experiments conducted under flow in the presence of RBC at physiologic hematocrit and shear rate, are a step forward in the prediction of in vivo cell&ndash;particle association. This approach has the potential to assist development and high-throughput screening of new endothelium-targeted nanocarriers

Endothelial Cell Targeting by cRGD-Functionalized Polymeric Nanoparticles under Static and Flow Conditions

Since αvβ3 integrin is a key component of angiogenesis in health and disease, Arg-Gly-Asp (RGD) peptide-functionalized nanocarriers have been investigated as vehicles for targeted delivery of drugs to the αvβ3 integrin-overexpressing neovasculature of tumors. In this work, PEGylated nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA) functionalized with cyclic-RGD (cRGD), were evaluated as nanocarriers for the targeting of angiogenic endothelium. For this purpose, NPs (~300 nm) functionalized with cRGD with different surface densities were prepared by maleimide-thiol chemistry and their interactions with human umbilical vein endothelial cells (HUVECs) were evaluated under different conditions using flow cytometry and microscopy. The cell association of cRGD-NPs under static conditions was time-, concentration- and cRGD density-dependent. The interactions between HUVECs and cRGD-NPs dispersed in cell culture medium under flow conditions were also time- and cRGD density-dependent. When washed red blood cells (RBCs) were added to the medium, a 3 to 8-fold increase in NPs association to HUVECs was observed. Moreover, experiments conducted under flow in the presence of RBC at physiologic hematocrit and shear rate, are a step forward in the prediction of in vivo cell-particle association. This approach has the potential to assist development and high-throughput screening of new endothelium-targeted nanocarriers