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Biomedical subjects

Timo L M ten Hagen

Publications and source records attributed to Timo L M ten Hagen.

At least 19 recordsLinked to original sources
PubMed2006

Differential effects of matrix and growth factors on endothelial and fibroblast motility: application of a modified cell migration assay.

Cell migration is crucial in virtually every biological process and strongly depends on the nature of the surrounding matrix. An assay that enables real-time studies on the effects of defined matrix components and growth factors on cell migration is not available. We have set up a novel, quantitative migration assay, which enables unharmed cells to migrate along a defined matrix. Here, we used this so-called barrier-assay to define the contribution of fibronectin (FN) and Collagen-I (Col-I) to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and lysophosphatidic acid (LPA)-induced cell migration of endothelial cells (EC) and fibroblasts. In EC, both FN and Col-I stimulated migration, but FN-induced motility was random, while net movement was inhibited. Addition of bFGF and VEGF overcame the effect of FN, with VEGF causing directional movement. In contrast, in 3T3 fibroblasts, FN stimulated motility and this effect was enhanced by bFGF. This motility was more efficient and morphologically completely different compared to LPA stimulation. Strikingly, directional migration of EC was not paralleled by higher amounts of stable microtubules (MT) or an increased reorientation of the microtubule-organizing centre (MTOC). For EC, the FN effect appeared concentration dependent; high FN was able to induce migration, while for fibroblasts both low and high concentrations of FN induced motility. Besides showing distinct responses of the different cells to the same factors, these results address contradictive reports on FN and show that the interplay between matrix components and growth factors determines both pattern and regulation of cell migration. J. Cell. Biochem. 99: 1536-1552, 2006. (c) 2006 Wiley-Liss, Inc.

3T3 Cells↗
PubMed2006

Synergistic antitumor effects of histamine plus melphalan in isolated hepatic perfusion for liver metastases.

BACKGROUND: Nonresectable primary and metastatic liver tumors remain an important clinical problem. Melphalan-based isolated hepatic perfusion (M-IHP) leads to more than 70% objective responses in selective groups of patients with nonresectable metastases confined to the liver. Complete responses are rare and progression-free survival is limited. Tumor necrosis factor (TNF), a very active agent in isolated limb perfusion, is linked to serious hepatotoxicity, restricting its use in IHP. Because of its vasoactive properties, histamine (Hi) is an alternative to TNF. In this article we evaluate its potential synergistic effect in M-IHP, improving response rates. METHODS: Our experimental rat IHP model is used for the treatment of soft tissue sarcoma liver metastases. Blood samples are collected for monitoring liver enzymes. Livers are excised 72 h and 7 days after treatment for histologic evaluation. RESULTS: After sham-IHP and Hi-IHP, tumor progression was observed in 100% of treated animals, while after M-IHP this number fell to 62% and after Hi + M-IHP it fell to only 22% (P = 0.006). Overall response rates were of 55% for Hi + M-IHP vs. 25% for M-IHP, and, more importantly, complete responses (CR) were observed only after Hi + M-IHP (22%) (P = 0.009). Hepatotoxicity peaked within 24 h after IHP, independent of the treatment administered, recovered in 48 h, and was related mainly to the elevation of transaminases (grade 3 ASAT and grade 1 ALAT for control group and grades 3 and 4, respectively, for all other treatments). No serious systemic toxicity was observed. Histology of the liver showed no serious damage. CONCLUSION: Hi + M-IHP has synergistic antitumor effects without any increase in regional or systemic toxicity.

Animals↗
PubMed2006

Endothelial monocyte-activating polypeptide-II and its functions in (patho)physiological processes.

Endothelial monocyte-activating polypeptide-II (EMAP-II) is a pro-inflammatory cytokine with anti-angiogenic properties. Its precursor, proEMAP, is identical to the p43 auxiliary component of the tRNA multisynthetase complex and therefore involved in protein translation. Although most of the activities have been ascribed to the active form EMAP-II, also p43 has reported cytokine properties. ProEMAP/p43 and EMAP-II act on many levels and on many cell types including endothelial cells, immune cells and fibroblasts. In this review we summarize all available data on isolation, expression and functions of EMAP-II both in physiological processes as well as in pathological settings, like cancer. We also discuss the different reported mechanisms for processing of proEMAP/p43 into EMAP-II. Finally, we speculate on the possible applications of this cytokine for (cancer) therapy.

Amino Acid Sequence↗
PubMed2006

Decreased response rates by the combination of histamine and IL-2 in melphalan-based isolated limb perfusion.

Histamine (Hi) combined to melphalan in a rat experimental model of isolated limb perfusion (ILP) for lower limb soft tissue sarcoma, resulted in overall response rates (OR) of 66%. Likewise, ILP with interleukin-2 (IL-2) resulted in OR of 67%, when combined to melphalan, in the same experimental model. In systemic immunotherapy, the combination of IL-2 and Hi has been used for solid tumor treatment based on immunomodulatory effects. In this study, we used our well-established ILP experimental model to evaluate whether the synergistic effect between the two drugs seen in the systemic setting, could further improve response rates in a loco-regional setting. Histological evaluation was done directly and 24 h after ILP. Melphalan uptake by tumor and muscle were measured. Hi and IL-2 together, combined to melphalan in the ILP led to OR of only 28%. Histology of tumors demonstrated partial loss of Hi-induced hemorrhagic effect when IL-2 was present. Melphalan accumulation in the tumor when both Hi and IL-2 were added (3.1-fold) was very similar to accumulation with Hi only (2.8-fold), or IL-2 only (3.5-fold) combined to melphalan. In vitro there was no synergy between the drugs. In conclusion there was a negative synergistic effect between IL-2 and Hi in the regional setting.

Animals↗
PubMed2006

Targeting of angiogenic endothelial cells at sites of inflammation by dexamethasone phosphate-containing RGD peptide liposomes inhibits experimental arthritis.

OBJECTIVE: To investigate whether RGD peptide-exposing long circulating polyethylene glycol (PEG) liposomes (RGD-PEG-L) targeted to alphavbeta3 integrins expressed on angiogenic vascular endothelial cells (VECs) are able to bind VECs at sites of inflammation and whether such liposomes containing dexamethasone phosphate (DEXP) can be used as carriers to interfere with the development of experimental arthritis. METHODS: Binding and internalization of RGD-PEG-L were studied by fluorescence-activated cell sorting and confocal microscopy using fluorescently labeled liposomes. Radiolabeled liposomes were used to test in vivo pharmacokinetics and inflammation site targeting in lipopolysaccharide (LPS)-induced inflammation and adjuvant-induced arthritis (AIA) in rats. In vivo inflammation targeting was visualized by intravital microscopy using fluorescently labeled RGD-PEG-L. Therapeutic efficacy of DEXP-encapsulating RGD-PEG-L compared with nontargeted liposomes was evaluated in rats with AIA. RESULTS: RGD-PEG-L bound to and were taken up by proliferating human VECs in vitro. In vivo, increased targeting of radiolabeled RGD-PEG-L to areas of LPS-induced inflammation in rats was observed. Specific association with the blood vessel wall at the site of inflammation was confirmed by intravital microscopy. One single intravenous injection of DEXP encapsulated in RGD-PEG-L resulted in a strong and long-lasting antiarthritic effect in rat AIA. CONCLUSION: RGD-targeted PEG liposomes represent an endothelial cell-specific drug delivery system that targets VECs at sites of inflammation. Use of these liposomes to deliver DEXP to VECs at arthritis-affected sites proved efficacious in rat adjuvant arthritis. These data indicate that VECs have an essential role in the inflammation process and suggest the possibility of using VEC targeting for therapeutic intervention in inflammatory processes such as arthritis.

Animals↗
PubMed2006

EMAP-II facilitates TNF-R1 apoptotic signalling in endothelial cells and induces TRADD mobilization.

Endothelial monocyte-activating polypeptide-II (EMAP-II), a proinflammatory cytokine with antiangiogenic properties, renders tumours sensitive to tumour necrosis factor-alpha (TNF) treatment. The exact mechanisms for this effect remain unclear. Here we show that human endothelial cells (EC) are insensitive to TNF-induced apoptosis but after a short pre-treatment with EMAP-II, EC quickly undergo TNF-induced apoptosis. We further analysed this EMAP-II pre-treatment effect and found no increase of TNF-R1 protein expression but rather an induction of TNF-R1 redistribution from Golgi storage pools to cell membranes. In addition, we observed EMAP-II induced mobilization and membrane expression of the TNF-R1-Associated Death Domain (TRADD) protein. Immunofluorescence co-staining experiments revealed that these two effects occurred at the same time in the same cell but TNF-R1 and TRADD were localized in different vesicles. These findings suggest that EMAP-II sensitises EC to apoptosis by facilitating TNF-R1 apoptotic signalling via TRADD mobilization and introduce a molecular and antiangiogenic explanation for the TNF sensitising properties of EMAP-II in tumours.

Apoptosis↗
PubMed2006

In vivo micro particle image velocimetry measurements of blood-plasma in the embryonic avian heart.

The measurement of blood-plasma velocity distributions with spatial and temporal resolution in vivo is inevitable for the determination of shear stress distributions in complex geometries at unsteady flow conditions like in the beating heart. A non-intrusive, whole-field velocity measurement technique is required that is capable of measuring instantaneous flow fields at sub-millimeter scales in highly unsteady flows. Micro particle image velocimetry (muPIV) meets these demands, but requires special consideration and methodologies in order to be utilized for in vivo studies in medical and biological research. We adapt muPIV to measure the blood-plasma velocity in the beating heart of a chicken embryo. In the current work, bio-inert, fluorescent liposomes with a nominal diameter of 400 nm are added to the flow as a tracer. Because of their small dimension and neutral buoyancy the liposomes closely follow the movement of the blood-plasma and allow the determination of the velocity gradient close to the wall. The measurements quantitatively resolve the velocity distribution in the developing ventricle and atrium of the embryo at nine different stages within the cardiac cycle. Up to 400 velocity vectors per measurement give detailed insight into the fluid dynamics of the primitive beating heart. A rapid peristaltic contraction accelerates the flow to peak velocities of 26 mm/s, with the velocity distribution showing a distinct asymmetrical profile in the highly curved section of the outflow tract. In relation to earlier published gene-expression experiments, the results underline the significance of fluid forces for embryonic cardiogenesis. In general, the measurements demonstrate that muPIV has the potential to develop into a general tool for instationary flow conditions in complex flow geometries encountered in cardiovascular research.

Animals↗
PubMed2006

Technology insight: Utility of TNF-alpha-based isolated limb perfusion to avoid amputation of irresectable tumors of the extremities.

Isolated limb perfusion (ILP) with melphalan is effective in the treatment of small multiple melanoma intransit metastases and is utilized widely for this indication. The treatment is much less effective against bulky melanoma metastases and has uniformly failed in the treatment of irresectable extremity soft tissue sarcomas. The addition of tumor-necrosis factor-alpha (TNF-alpha) to this treatment approach has changed the situation dramatically. High response rates and limb-salvage rates have been reported in multicenter trials that combined ILP with TNF-alpha plus melphalan; these trials resulted in the approval of TNF-alpha for bulky melanoma metastases and soft tissue sarcomas in Europe in 1998. Subsequently, many doctors working in European centers have been trained, and a series of confirmatory reports from single institutions have now been published regarding the efficacy of the procedure. TNF-alpha has an early and a late effect; it enhances tumor-selective drug uptake during the perfusion, and plays an essential role in the subsequent selective destruction of the tumor vasculature. These effects result in a high response rate in bulky tumors, soft tissue sarcomas, bulky melanomas, and various other tumor types. This induction therapy therefore allows tumor remnants to be resected some 3 months after ILP thus avoiding limb amputation. TNF-alpha-based ILP is a well-established treatment that aims to avoid amputations regardless of the tumor size and type. It represents an important example of combination therapy that modulates the tumor vasculature and should be offered in high-volume tertiary referral centers.

Amputation, Surgical↗
PubMed2006

Changing the pathophysiology of solid tumours: the potential of TNF and other vasoactive agents.

Solid tumour therapy with chemotherapeutic drugs is hampered by a number of factors resulting in poor results and failure of initially promising drugs. From the application of Tumour Necrosis Factor alpha in the melphalan-based Isolated Limb perfusion some lessons could be learned. Most importantly, combination of treatment approaches, certainly when multiple targets are involved, increases the effectiveness of the therapy. Clinical outcome may improve dramatically when the tumour pathophysiology is changed in such a way that co-administered chemotherapeutics are more active or are capable of reaching the tumour cells better. Here some of the methodologies and drug combinations which improve solid tumour therapy through acting on the tumour pathophysiology are discussed.

Antineoplastic Agents↗
PubMed2006

Early destruction of tumor vasculature in tumor necrosis factor-alpha-based isolated limb perfusion is responsible for tumor response.

Addition of high-dose tumor necrosis factor-alpha to melphalan-based isolated limb perfusion enhances anti-tumor effects impressively. Unfortunately, the mechanism of action of tumor necrosis factor-alpha is still not fully understood. Here, we investigated the effects of tumor necrosis factor-alpha on the tumor microenvironment and on secondary immunological events during and shortly after isolated limb perfusion in soft-tissue sarcoma-bearing rats. Already during isolated limb perfusion, softening of the tumor was observed. Co-administration of tumor necrosis factor-alpha in the isolated limb perfusion with melphalan induced a six-fold enhanced drug accumulation of melphalan in the tumor compared with isolated limb perfusion with melphalan alone. In addition, directly after perfusion with tumor necrosis factor-alpha plus melphalan, over a time-frame of 30 min, vascular destruction, erythrocyte extravasation and hemorrhage was detected. Interstitial fluid pressure and pH in the tumor, however, were not altered by tumor necrosis factor-alpha and no clear immune effects, cellular infiltration or cytokine expression were observed. Taken together, these results indicate that tumor necrosis factor-alpha induces rapid damage to the tumor vascular endothelial lining resulting in augmented drug accumulation. As other important parameters were not changed (e.g. interstitial fluid pressure and pH), we speculate that the tumor vascular changes, and concurrent hemorrhage and drug accumulation are the key explanations for the observed synergistic anti-tumor response.

Animals↗
PubMed2005

Synergistic antitumor response of interleukin 2 with melphalan in isolated limb perfusion in soft tissue sarcoma-bearing rats.

The cytokine interleukin 2 (IL-2) is a mediator of immune cell activation with some antitumor activity, mainly in renal cell cancer and melanoma. We have previously shown that tumor necrosis factor (TNF)-alpha has strong synergistic antitumor activity in combination with chemotherapeutics in the isolated limb perfusion (ILP) setting based on a TNF-mediated enhanced tumor-selective uptake of the chemotherapeutic drug followed by a selective destruction of the tumor vasculature. IL-2 can cause vascular leakage and edema and for this reason we examined the antitumor activity of a combined treatment with IL-2 and melphalan in our well-established ILP in soft tissue sarcoma-bearing rats (BN175). ILP with either IL-2 or melphalan alone has no antitumor effect, but the combination of IL-2 and melphalan resulted in a strong synergistic tumor response, without any local or systemic toxicity. IL-2 enhanced significantly melphalan uptake in tumor tissue. No signs of significant vascular damage were detected to account for this observation, although the tumor sections of the IL-2- and IL-2 plus melphalan-treated animals revealed scattered extravasation of erythrocytes compared with the untreated animals. Clear differences were seen in the localization of ED-1 cells, with an even distribution in the sham, IL-2 and melphalan treatments, whereas in the IL-2 plus melphalan-treated tumors clustered ED-1 cells were found. Additionally, increased levels of TNF mRNA were found in tumors treated with IL-2 and IL-2 plus melphalan. These observations indicate a potentially important role for macrophages in the IL-2-based perfusion. The results in our study indicate that the novel combination of IL-2 and melphalan in ILP has synergistic antitumor activity and may be an alternative for ILP with TNF and melphalan.

Animals↗
PubMed2005

Liposomal cytokines in the treatment of infectious diseases and cancer.

Despite of the demonstrated activity of cytokines in vitro, their use in the clinical setting is often disappointing. Cytokine-related toxicity seriously limits optimal use in vivo. In addition, rapid degradation and excretion, neutralization and binding to receptors, or metabolization of the molecule results in a short half-life in serum when injected intravenously. As the dose-response curve of cytokines is relatively steep, outcome greatly benefits from improved delivery and bioavailability. One way to improve the pharmacokinetics of cytokines after systemic application is encapsulation in liposomes. An advantage of liposomes is that the encapsulated drug is protected from (rapid) degradation and excretion, and it eliminates the binding to neutralizing antibodies or (soluble) receptors. Moreover, liposomes can be tailored in such a way that they exhibit favorable pharmacokinetics, i.e., increased serum half-life and improved targeting to tissues or cells of interest. In this chapter, the use of liposomal cytokines in the treatment of cancer and infectious disease is discussed.

Animals↗
PubMed2005

Addition of low-dose tumor necrosis factor-alpha to systemic treatment with STEALTH liposomal doxorubicin (Doxil) improved anti-tumor activity in osteosarcoma-bearing rats.

Improved efficacy of Doxil (STEALTH liposomal doxorubicin) compared to free doxorubicin has been demonstrated in the treatment of several tumor types. We have shown that addition of low-dose tumor necrosis factor (TNF) to systemic Doxil administration dramatically improved tumor response in the highly vascularized rat soft tissue sarcoma BN175. Whether a similar enhanced efficacy can be achieved in less vascularized tumors is uncertain. We therefore examined the effect of systemic administration of Doxil in combination with low-dose TNF in intermediate vascularized osteosarcoma-bearing rats (ROS-1). Small fragments of the osteosarcoma were implanted s.c. in the lower limb. Treatment was started when the tumors reached an average diameter of 1 cm. Rats were treated with five i.v. injections at 4-day intervals with Doxil or doxorubicin and TNF. Systemic treatment with Doxil resulted in a better tumor growth delay than free doxorubicin, but with progressive diseases in all animals. The 3.5-fold augmented accumulation of Doxil compared to free doxorubicin presumably explains the enhanced tumor regression. Addition of low-dose TNF augmented the anti-tumor activity of Doxil, although no increased drug uptake was found compared to Doxil alone. In vitro studies showed that ROS-1 is sensitive to TNF, but systemic treatment with TNF alone did not result in a tumor growth delay. Furthermore, we demonstrated that treatment with Doxil alone or with TNF resulted in massive coagulative necrosis of tumor tissue. In conclusion, combination therapy of Doxil and low-dose TNF seems attractive for the treatment of highly vascularized tumors, but also of intermediate vascularized tumors like the osteosarcoma.

Animals↗
PubMed2005

MR angiography of tumor-related vasculature: from the clinic to the micro-environment.

Angiogenesis is a very important process for tumor growth and proliferation. Given its high temporal and spatial resolution, magnetic resonance (MR) imaging is well suited for use in the assessment of angiogenesis. MR angiography can be used clinically and experimentally for identification of tumor feeding and draining vessels, for tumor characterization, and for treatment planning. The morphologic structure of tumor vessels can be investigated in relation to tumor vessel permeability with use of specific contrast agents. To gain insight into tumor angiogenesis in vivo, the authors compared images obtained with digital photography, high-resolution MR angiography, and intravital microscopy through a dorsal skin-fold window in a rodent model. The close correlation between images obtained with these various modalities, with regard to the depiction of the developing tumor vasculature, indicates that noninvasive quantification of angiogenesis may be possible with MR imaging. Future directions in tumor imaging may include so-called four-dimensional MR angiography, in which high-resolution three-dimensional MR angiography is combined with dynamic contrast-enhanced MR imaging.

Animals↗
PubMed2005

Gene therapy in in vivo isolated perfusion models.

Locoregional administration of a genetic construct by means of in vivo, in situ isolated perfusion (IP) of a target organ or extremity is a method that may increase in vivo efficacy. Vascular isolation and perfusion minimizes systemic exposure and thereby reduces unwanted side effects. Isolated hepatic perfusion (IHP) is the most extensively studied IP model, especially in gene therapy protocols for inborn errors of metabolism. To achieve stable transduction most frequently retroviruses are used in IHP. IHP is combined with hepatectomy or vascular ligation of liver lobes to induce liver regeneration increasing transduction efficacy. When adenoviruses are used in IHP high transduction percentages of hepatocytes can be achieved without significant toxicity. In tumor models adenoviral IHP has been performed, but has not been very successful up till now. Isolated limb perfusion (ILP) is a promising treatment modality in pre-clinical cancer gene therapy studies. After ILP a homogeneous distribution of transduced cells was demonstrated especially at the viable rim of the tumor and around tumor associated vessels. Moreover complete tumor responses have been observed. Isolated pulmonary perfusion (IPP) results in selective expression in the perfused lung and the duration of expression is longer than after systemic administration. In rats a significant decrease of tumor nodules upon IPP can be achieved. Furthermore other less studied perfusion models are discussed: isolated kidney perfusion (IKP), isolated spleen perfusion (ISP) and isolated cardiac perfusion (ICP). IP is a methodology that delivers vectors highly selectively, with a long exposure time and high concentrations at the target side. This results in higher transduction rates and thereby may improve therapeutic effects.

Animals↗
PubMed2004

Synergistic antitumor activity of histamine plus melphalan in isolated limb perfusion: preclinical studies.

BACKGROUND: We have previously shown how tumor response of isolated limb perfusion (ILP) with melphalan was improved when tumor necrosis factor alpha (TNF-alpha) was added. Taking into account that other vasoactive drugs could also improve tumor response to ILP, we evaluated histamine (Hi) as an alternative to TNF-alpha. METHODS: We used a rat ILP model to assess the combined effects of Hi and melphalan (n = 6) on tumor regression, melphalan uptake (n = 6), and tissue histology (n = 2) compared with Hi or melphalan alone. We also evaluated the growth of BN-175 tumor cells as well as apoptosis, necrosis, cell morphology, and paracellular permeability of human umbilical vein endothelial cells (HUVECs) after Hi treatment alone and in combination with melphalan. RESULTS: The antitumor effect of the combination of Hi and melphalan in vivo was synergistic, and Hi-dependent reduction in tumor volume was blocked by H1 and H2 receptor inhibitors. Tumor regression was observed in 66% of the animals treated with Hi and melphalan, compared with 17% after treatment with Hi or melphalan alone. Tumor melphalan uptake increased and vascular integrity in the surrounding tissue was reduced after ILP treatment with Hi and melphalan compared with melphalan alone. In vitro results paralleled in vivo results. BN-175 tumor cells were more sensitive to the cytotoxicity of combined treatment than HUVECs, and Hi treatment increased the permeability of HUVECs. CONCLUSIONS: Hi in combination with melphalan in ILP improved response to that of melphalan alone through direct and indirect mechanisms. These results warrant further evaluation in the clinical ILP setting and, importantly, in organ perfusion.

Animals↗
PubMed2004

Tumor necrosis factor-alpha augmented tumor response in B16BL6 melanoma-bearing mice treated with stealth liposomal doxorubicin (Doxil) correlates with altered Doxil pharmacokinetics.

The application of tumor necrosis factor-alpha (TNF) for the treatment of solid tumors is limited by its severe, life-threatening, toxicity. Therefore, only low dosages of this cytokine can be applied systemically, which results in poor tumor response. It has been demonstrated previously that administration of high-dose TNF in a so-called isolated perfusion system markedly improved tumor response when combined with chemotherapy. It appeared that TNF had a major impact specifically on the tumor-associated vasculature. At these high concentrations, endothelial cell death is induced by TNF, resulting in complete collapse of the tumor vascular bed. Strikingly, this effect alone is not enough to induce a tumor response, but addition of a chemotherapeutic drug is mandatory to obtain an anti-tumor effect. We showed that TNF has no anti-tumor effect by itself but augmented drug accumulation mainly in the tumor, most likely by enhancing vascular leakage. It seems that enhanced vascular leakage, but not endothelial cell death, explains the interaction between TNF and the co-administered drug. We hypothesized that in a low-dose setting TNF could induce tumor accumulation of chemotherapeutic drugs and consequently improve tumor response. We demonstrate that free TNF has a strong effect on the pharmacokinetics of co-administered Doxil in B16BL6 melanoma-bearing mice, resulting in strongly augmented drug accumulation in the tumor and improved tumor response. Co-injection of Stealth liposomal TNF with Doxil resulted in comparable or less pronounced tumor responses as compared to free TNF. These results imply that systemic application of clinically tolerable doses of TNF may improve drug distribution and tumor response and could be useful in a number of anti-cancer therapies.

Animals↗