A Three-Dimensional Bioabsorbable Tissue Marker for Volume Replacement and Radiation Planning: A Multicenter Study of Surgical and Patient-Reported Outcomes for 818 Patients with Breast Cancer.

BACKGROUND: Accurate identification of the tumor bed after breast-conserving surgery (BCS) ensures appropriate radiation to the tumor bed while minimizing normal tissue exposure. The BioZorb® three-dimensional (3D) bioabsorbable tissue marker provides a reliable target for radiation therapy (RT) planning and follow-up evaluation while serving as a scaffold to maintain breast contour. METHODS: After informed consent, 818 patients (826 breasts) implanted with the BioZorb® at 14 U.S. sites were enrolled in a national registry. All the patients were prospectively followed with the BioZorb® implant after BCS. The data collected at 3, 6, 12, and 24 months included all demographics, treatment parameters, and provider/patient-assessed cosmesis. RESULTS: The median follow-up period was 18.2 months (range, 0.2-53.4 months). The 30-day breast infection rate was 0.5 % of the patients (n = 4), and re-excision was performed for 8.1 % of the patients (n = 66), whereas 2.6 % of the patients (n = 21) underwent mastectomy. Two patients (0.2 %) had local recurrence. The patient-reported cosmetic outcomes at 6, 12, and 24 months were rated as good-to-excellent by 92.4 %, 90.6 %, and 87.3 % of the patients, respectively and similarly by the surgeons. The radiation oncologists reported planning of target volume (PTV) reduction for 46.2 % of the patients receiving radiation boost, with PTV reduction most commonly estimated at 30 %. CONCLUSIONS: This report describes the first large multicenter study of 818 patients implanted with the BioZorb® tissue marker during BCS. Radiation oncologists found that the device yielded reduced PTVs and that both the patients and the surgeons reported good-to-excellent long-term cosmetic outcomes, with low adverse effects. The BioZorb® 3D tissue marker is a safe adjunct to BCS and may add benefits for both surgeons and radiation oncologists.

Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells.

The MEKK3/MEK5/ERK5 signaling axis is required for cardiovascular development in vivo. We analyzed the physiological role of ERK5 in cardiac endothelial cells and the consequence of activation of this kinase by the statin class of HMG Co-A reductase inhibitor drugs. We utilized human cardiac microvascular endothelial cells (HCMECs) and altered ERK5 expression using siRNA mediated gene silencing or overexpression of constitutively active MEK5 and ERK5 to reveal a role for ERK5 in regulating endothelial tight junction formation and cell permeability. Statin treatment of HCMECs stimulated activation of ERK5 and translocation to the plasma membrane resulting in co-localization with the tight junction protein ZO-1 and a concomitant reduction in endothelial cell permeability. Statin mediated activation of ERK5 was a consequence of reduced isoprenoid synthesis following HMG Co-A reductase inhibition. Statin pretreatment could overcome the effect of doxorubicin in reducing endothelial tight junction formation and prevent increased permeability. Our data provide the first evidence for the role of ERK5 in regulating endothelial tight junction formation and endothelial cell permeability. Statin mediated ERK5 activation and the resulting decrease in cardiac endothelial cell permeability may contribute to the cardioprotective effects of statins in reducing doxorubicin-induced cardiotoxicity.

Cellular Uptake of the Atypical Antipsychotic Clozapine Is a Carrier-Mediated Process.

The weak base antipsychotic clozapine is the most effective medication for treating refractory schizophrenia. The brain-to-plasma concentration of unbound clozapine is greater than unity, indicating transporter-mediated uptake, which has been insufficiently studied. This is important, because it could have a significant impact on clozapine's efficacy, drug-drug interaction, and safety profile. A major limitation of clozapine's use is the risk of clozapine-induced agranulocytosis/granulocytopenia (CIAG), which is a rare but severe hematological adverse drug reaction. We first studied the uptake of clozapine into human brain endothelial cells (hCMEC/D3). Clozapine uptake into cells was consistent with a carrier-mediated process, which was time-dependent and saturable ( Vmax = 3299 pmol/million cells/min, Km = 35.9 µM). The chemical inhibitors lamotrigine, quetiapine, olanzapine, prazosin, verapamil, indatraline, and chlorpromazine reduced the uptake of clozapine by up to 95%. This could in part explain the in vivo interactions observed in rodents or humans for these compounds. An extensive set of studies utilizing transporter-overexpressing cell lines and siRNA-mediated transporter knockdown in hCMEC/D3 cells showed that clozapine was not a substrate of OCT1 (SLC22A1), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), ENT1 (SLC29A1), ENT2 (SLC29A2), and ENT4/PMAT (SLC29A4). In a recent genome-wide analysis, the hepatic uptake transporters SLCO1B1 (OATP1B1) and SLCO1B3 (OATP1B3) were identified as additional candidate transporters. We therefore also investigated clozapine transport into OATP1B-transfected cells and found that clozapine was neither a substrate nor an inhibitor of OATP1B1 and OATP1B3. In summary, we have identified a carrier-mediated process for clozapine uptake into brain, which may be partly responsible for clozapine's high unbound accumulation in the brain and its drug-drug interaction profile. Cellular clozapine uptake is independent from currently known drug transporters, and thus, molecular identification of the clozapine transporter will help to understand clozapine's efficacy and safety profile.

RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

Impact of a Novel Bioabsorbable Implant on Radiation Treatment Planning for Breast Cancer.

BACKGROUND: Techniques for accurately delineating the tumor bed after breast-conserving surgery (BCS) can be challenging. As a result, the accuracy, and efficiency of radiation treatment (RT) planning can be negatively impacted. Surgically placed clips or the post-surgical seroma are commonly used to determine target volume; however, these methods can lead to a high degree of uncertainty and variability. A novel 3-dimensional bioabsorbable marker was used during BCS and assessed for its impact on RT planning. METHODS: One hundred and ten implants were sutured to the margins of the tumor bed excision site in 108 patients undergoing BCS. Routine CT imaging of the breast tissue was performed for RT planning, and the marker was assessed for visibility and utility in target delineation. RT regimens, target volumes and associated treatment costs were analyzed. RESULTS: In all patients, the marker was easily visible and in 95.7 % of cases, it proved useful for RT planning. 36.8 % of patients received conventional whole breast irradiation plus boost, 56.6 % received hypo-fractionation plus boost, and 6.6 % received accelerated partial breast irradiation. A shift toward increased use of hypo-fractionated regimens was noted over the three year period of this study. There were no device-related complications or cancer recurrences in this group of patients. CONCLUSIONS: This study demonstrated the use of a novel 3-dimensional marker as a safe and effective method for delineating the tumor bed with a significant utility for RT planning. With routine use of the device, an increased use of hypofractionation with a resultant 25 % cost savings was noted.

The role of ERK5 in endothelial cell function.

Extracellular-signal-regulated kinase 5 (ERK5), also termed big MAPK1 (BMK1), is the most recently discovered member of the mitogen-activated protein kinase (MAPK) family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that, in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling and regulating tumour angiogenesis. The present review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.

VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

Extracellular-signal-regulated kinase 5 (ERK5) is critical for normal cardiovascular development. Previous studies have defined a canonical pathway for ERK5 activation, showing that ligand stimulation leads to MEK5 activation resulting in dual phosphorylation of ERK5 on Thr218/Tyr220 residues within the activation loop. ERK5 then undergoes a conformational change, facilitating phosphorylation on residues in the C-terminal domain and translocation to the nucleus where it regulates MEF2 transcriptional activity. Our previous research into the importance of ERK5 in endothelial cells highlighted its role in VEGF-mediated tubular morphogenesis and cell survival, suggesting that ERK5 played a unique role in endothelial cells. Our current data show that in contrast to EGF-stimulated HeLa cells, VEGF-mediated ERK5 activation in human dermal microvascular endothelial cells (HDMECs) does not result in C-terminal phosphorylation of ERK5 and translocation to the nucleus, but instead to a more plasma membrane/cytoplasmic localisation. Furthermore, the use of small-molecule inhibitors to MEK5 and ERK5 shows that instead of regulating MEF2 activity, VEGF-mediated ERK5 is important for regulating AKT activity. Our data define a novel pathway for ERK5 activation in endothelial cells leading to cell survival.

Mesothelial Cells Exhibit Characteristics of Perivascular Cells in an In Vitro Angiogenesis Assay.

Mesothelial cells have been shown to have remarkable plasticity towards mesenchymal cell types during development and in disease situations. Here, we have characterized the potential of mesothelial cells to undergo changes toward perivascular cells using an in vitro angiogenesis assay. We demonstrate that GFP-labeled mesothelial cells (GFP-MCs) aligned closely and specifically with endothelial networks formed when human dermal microvascular endothelial cells (HDMECs) were cultured in the presence of VEGF-A165 on normal human dermal fibroblasts (NHDFs) for a 7-day period. The co-culture with GFP-MCs had a positive effect on branch point formation indicating that the cells supported endothelial tube formation. We interrogated the molecular response of the GFP-MCs to the angiogenic co-culture by qRT-PCR and found that the pericyte marker Ng2 was upregulated when the cells were co-cultured with HDMECs on NHDFs, indicating a change towards a perivascular phenotype. When GFP-MCs were cultured on the NHDF feeder layer, they upregulated the epithelial-mesenchymal transition marker Zeb1 and lost their circularity while increasing their size, indicating a change to a more migratory cell type. We analyzed the pericyte-like behavior of the GFP-MCs in a 3D cardiac microtissue (spheroid) with cardiomyocytes, cardiac fibroblasts and cardiac endothelial cells where the mesothelial cells showed alignment with the endothelial cells. These results indicate that mesothelial cells have the potential to adopt a perivascular phenotype and associate with endothelial cells to potentially support angiogenesis.

Umbilical cord mesenchymal stromal cell-derived extracellular vesicles lack the potency to immunomodulate human monocyte-derived macrophages in vitro.

Mesenchymal stromal cells (MSCs) have been reported to display efficacy in a variety of preclinical models, but without long-term engraftment, suggesting a role for secreted factors, such as MSC-derived extracellular vesicles (EVs). MSCs are known to elicit immunomodulatory effects, an important aspect of which is their ability to affect macrophage phenotype. However, it is not clear if these effects are mediated by MSC-derived EVs, or other factors secreted by the MSCs. Here, we use flow cytometry to assess the effects of human umbilical cord (hUC) MSC-derived EVs on the expression of pro-inflammatory (CD80) and anti-inflammatory (CD163) surface markers in human monocyte-derived macrophages (hMDMs). hUC-MSC-derived EVs did not change the surface marker expression of the hMDMs. In contrast, when hMDMs were co-incubated with hUC-MSCs in indirect co-cultures, changes were observed in the expression of CD14, CD80 and CD163, particularly in M1 macrophages, suggesting that soluble factors are necessary to elicit a shift in phenotype. However, even though EVs did not alter the surface marker expression of macrophages, they promoted angiogenesis and phagocytic capacity increased proportionally to increases in EV concentration. Taken together, these results suggest that hUC-MSC-derived EVs are not sufficient to alter macrophage phenotype and that additional MSC-derived factors are needed.

Exogenous recombinant dimeric neuropilin-1 is sufficient to drive angiogenesis.

Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(121), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells.

ERK5 is required for VEGF-mediated survival and tubular morphogenesis of primary human microvascular endothelial cells.

Extracellular signal-regulated kinase 5 (ERK5) is activated in response to environmental stress and growth factors. Gene ablation of Erk5 in mice is embryonically lethal as a result of disruption of cardiovascular development and vascular integrity. We investigated vascular endothelial growth factor (VEGF)-mediated ERK5 activation in primary human dermal microvascular endothelial cells (HDMECs) undergoing proliferation on a gelatin matrix, and tubular morphogenesis within a collagen gel matrix. VEGF induced sustained ERK5 activation on both matrices. However, manipulation of ERK5 activity by siRNA-mediated gene silencing disrupted tubular morphogenesis without impacting proliferation. Overexpression of constitutively active MEK5 and ERK5 stimulated tubular morphogenesis in the absence of VEGF. Analysis of intracellular signalling revealed that ERK5 regulated AKT phosphorylation. On a collagen gel, ERK5 regulated VEGF-mediated phosphorylation of the pro-apoptotic protein BAD and increased expression of the anti-apoptotic protein BCL2, resulting in decreased caspase-3 activity and apoptosis suppression. Our findings suggest that ERK5 is required for AKT phosphorylation and cell survival and is crucial for endothelial cell differentiation in response to VEGF.

Extracellular Vesicles from Human Umbilical Cord-Derived MSCs Affect Vessel Formation In Vitro and Promote VEGFR2-Mediated Cell Survival.

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have emerged as novel tools in regenerative medicine. Angiogenesis modulation is widely studied for the treatment of ischaemic diseases, wound healing, and tissue regeneration. Here, we have shown that EVs from human umbilical cord-derived MSCs can affect VEGFR2 signalling, a master regulator of angiogenesis homeostasis, via altering the phosphorylation of AKT. This translates into an inhibition of apoptosis, promoting exclusively cell survival, but not proliferation, in human microvascular endothelial cells. Interestingly, when comparing EVs from normoxic cells to those obtained from hypoxia (1% O2) preconditioned cells, hypoxia-derived EVs appear to have a slightly enhanced effect. Furthermore, when studied in a longer term endothelial-fibroblast co-culture angiogenesis model in vitro, both EV populations demonstrated a positive effect on vessel formation, evidenced by increased vessel networks with tubes of significantly larger diameters. Our data reveals that EVs selectively target components of the angiogenic pathway, promoting VEGFR2-mediated cell survival via enhancement of AKT activation. Our data show that EVs are able to enhance specific components of the VEGF signalling pathway and may have therapeutic potential to support endothelial cell survival.

Inhibiting ERK5 overcomes breast cancer resistance to anti-HER2 therapy by targeting the G1/S cell cycle transition.

Targeting the human epidermal growth factor receptor 2 (HER2) became a landmark in the treatment of HER2-driven breast cancer. Nonetheless, the clinical efficacy of anti-HER2 therapies can be short-lived and a significant proportion of patients ultimately develop metastatic disease and die. One striking consequence of oncogenic activation of HER2 in breast cancer cells is the constitutive activation of the extracellular-regulated protein kinase 5 (ERK5) through its hyperphosphorylation. In this study, we sought to decipher the significance of this unique molecular signature in promoting therapeutic resistance to anti-HER2 agents. We found that a small-molecule inhibitor of ERK5 suppressed the phosphorylation of the retinoblastoma protein (RB) in HER2 positive breast cancer cells. As a result, ERK5 inhibition enhanced the anti-proliferative activity of single-agent anti-HER2 therapy in resistant breast cancer cell lines by causing a G1 cell cycle arrest. Moreover, ERK5 knockdown restored the anti-tumor activity of the anti-HER2 agent lapatinib in human breast cancer xenografts. Taken together, these findings support the therapeutic potential of ERK5 inhibitors to improve the clinical benefit that patients receive from targeted HER2 therapies.

Transcriptional profiling reveals a critical role for tyrosine phosphatase VE-PTP in regulation of VEGFR2 activity and endothelial cell morphogenesis.

To define molecular events accompanying formation of the 3-dimensional (3D) vascular tube, we have characterized gene expression during vascular endothelial growth factor (VEGF)-induced tubular morphogenesis of endothelial cells. Microarray analyses were performed comparing gene induction in growth-arrested, tube-forming endothelial cells harvested from 3D collagen cultures to that in proliferating endothelial cells cultured on fibronectin. Differentially expressed genes were clustered and analyzed for specific endothelial expression through publicly available datasets. We validated the contribution of one of the identified genes, vascular endothelial protein tyrosine phosphatase (VE-PTP), to endothelial morphogenesis. Silencing of VE-PTP expression was accompanied by increased VEGF receptor-2 (VEGFR2) tyrosine phosphorylation and activation of downstream signaling pathways. The increased VEGFR2 activity promoted endothelial cell cycle progression, overcoming the G(0)/G(1) arrest associated with organization into tubular structures in the 3D cultures. Proximity ligation showed close association between VEGFR2 and VE-PTP in resting cells. Activation of VEGFR2 by VEGF led to rapid loss of association, which was resumed with time in parallel with decreased receptor activity. In conclusion, we have identified genes, which may serve critical functions in formation of the vascular tube. One of these, VE-PTP, regulates VEGFR2 activity thereby modulating the VEGF-response during angiogenesis.

ERK5 and the regulation of endothelial cell function.

ERK5 (extracellular-signal-regulated kinase 5), also termed BMK1 [big MAPK1 (mitogen-activated protein kinase 1)], is the most recently discovered member of the MAPK family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling, regulating hypoxia, tumour angiogenesis and cell migration. This review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.

VEGF-receptor signal transduction.

The vascular endothelial growth factor (VEGF) family of ligands and receptors has been the focus of attention in vascular biology for more than a decade. There is now a consensus that the VEGFs are crucial for vascular development and neovascularization in physiological and pathological processes in both embryo and adult. This has facilitated a rapid transition to their use in clinical applications, for example, administration of VEGF ligands to enhance vascularization of ischaemic tissues and, conversely, inhibitors of VEGF-receptor function in anti-angiogenic therapy. More recent data indicate essential roles for the VEGFs in haematopoietic cell function and in lymphangiogenesis.

Attenuation of doxorubicin-induced cardiotoxicity in a human in vitro cardiac model by the induction of the NRF-2 pathway.

Dose-dependent cardiotoxicity is the leading adverse reaction seen in cancer patients treated with doxorubicin. Currently, dexrazoxane is the only approved drug that can partially protect against this toxicity in patients, however, its administration is restricted to those patients receiving a high cumulative dose of anthracyclines. Investigations into the mechanisms of cardiotoxicity and efforts to improve cardioprotective strategies have been hindered by the limited availability of a phenotypically relevant in vitro adult human cardiac model system. Here, we adapted a readily reproducible, functional 3D human multi-cell type cardiac system to emulate patient responses seen with doxorubicin and dexrazoxane. We show that administration of two NRF2 gene inducers namely the semi-synthetic triterpenoid Bardoxolone methyl, and the isothiocyanate sulfurophane, result in cardioprotection against doxorubicin toxicity comparable to dexrazoxane as evidenced by an increase in cell viability and a decrease in the production of reactive oxygen species. We further show a synergistic attenuation of cardiotoxicity when the NRF2 inducers and dexrazoxane are used in tandem. Taken together, our data indicate that the 3D spheroid is a suitable model to investigate drug induced cardiotoxicity and we reveal an essential role of the NRF2 pathway in cardioprotection providing a novel pharmacological mechanism and intervention route towards the alleviation of doxorubicin-induced toxicity.

Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition.

Vascular endothelial growth factors (VEGFs) regulate vascular development, angiogenesis and lymphangiogenesis by binding to a number of receptors. VEGFR-1 is required for the recruitment of haematopoietic stem cells and the migration of monocytes and macrophages, VEGFR-2 regulates vascular endothelial function and VEGFR-3 regulates lymphatic endothelial cell function. Over the last decade, considerable progress has been made in delineating the VEGFR-2 specific intracellular signalling cascades leading to proliferation, migration, survival and increased permeability, each of which contributes to the angiogenic response. Furthermore, therapeutic inhibition of VEGFR-2 action is now having an impact in the clinic for the treatment of a number of diseases.

The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-gamma (PLC-gamma). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-alpha and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-gamma activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1-mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-gamma activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1-mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1-mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.

Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability.

Cardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1) levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs) leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes.