Bruce M. Fenton

Nonuniform red cell distribution in 20 to 100 μm bifurcations

Several potentially important parameters influencing the disproportionate distribution of red cell flux and blood flow at a bifurcation are examined. These include parent vessel hematocrit, vessel diameter, suspending medium, cell distensibility, parent vessel blood flow rate, and local geometry. Measurements were performed on 20 to 100 micrometers bifurcations, fabricated such that all vessels of a given bifurcations have the same diameter. Suspensions of human red cells, hardened red cells, and mixtures of each in albumin-saline, Dextran 75, or plasma were flowed through the bifurcations and determinations of flow rates and discharge hematocrits were made for each of the channels. For the 20-micrometers channels, hematocrits were found using videophotometric techniques, and for the larger channels, hematocrits were measured directly from the exit streams. Flow rates for both were measured by meniscus travel downstream in small-diameter glass tubes. Within the limits of the present experiments, three of the variables proved to be of major importance: feed hematocrit, tube diameter, and flow-rate distribution. It was clearly demonstrated that red cell flux varies nonlinearly with fractional flow rate. Critical flow rates, at which all or none of the cells entered one of the branches, were found to vary with diameter and hematocrit as has been reported in other studies. The data were analyzed with a theoretical model which assumes that the parent vessel contains a core of uniformly distributed red cells surrounded by a marginal gap of suspending medium; in the parent vessel lumen, the flows to the two daughter branches were assumed to be separated by a chord. The marginal gap widths and tube hematocrits deduced from the data with this model are of reasonable magnitudes.

Effect of Administration of Lovastatin on the Development of Late Pulmonary Effects after Whole-Lung Irradiation in a Murine Model

Abstract Williams, J. P., Hernady, E., Johnston, C. J., Reed, C. M., Fenton, B., Okunieff, P. and Finkelstein, J. N. Effect of Administration of Lovastatin on the Development of Late Pulmonary Effects after Whole-Lung Irradiation in a Murine Model. Radiat. Res. 161, 560–567 (2004). Our groups work on late radiation effects has been governed by the hypothesis that the effects observed in normal tissues are a consequence of multicellular interactions through a network of mediators. Further, we believe that inflammation is a necessary component of this process. We therefore investigated whether the recruitment of mononuclear cells, observed during the pneumonitic period in the irradiated normal lung, is dependent on the expression of chemokines, notably Mcp1. Since statins have been shown to reduce chemokine expression and inflammatory cell recruitment, we specifically examined whether statins could be used to reduce monocyte recruitment. Mice received 15 Gy whole-lung irradiation; treated groups were administered lovastatin three times weekly starting either immediately or 8 weeks postirradiation. At subsequent intervals, animals were killed humanely, and cellular, mRNA and protein analyses were undertaken. Statin-treated animals demonstrated a statistically significant reduction in both macrophage and lymphocyte populations in the lung compared to radiation alone as well as improved rates of survival and decreased collagen content. In addition, ELISA measurements showed that radiation-induced increases in Mcp1 protein were reduced by statin treatment. Additional experiments are needed to assess whether statins offer a potential treatment for the amelioration of late effects in breast and lung cancer patients undergoing radiation therapy.

Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements

SummaryDespite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumour blocks. Second, hypoxic development is quantified in adjacent sections using immunohistochemical detection of a fluorescently conjugated monoclonal antibody (ELK3-51) to a nitroheterocyclic hypoxia marker (EF5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent (Hoechst 33342 or DiOC7(3)) and immunohistological (PECAM-1/CD31) stains is used to define the anatomical vascular densities and the fraction of blood vessels containing flow. Using a computer-interfaced microscope stage, image analysis software and a 3-CCD colour video camera, multiple images are digitized, combined to form a photo-montage and revisited after each of the three staining protocols. By applying image registration techniques, the spatial distribution of HbO2 saturations is matched to corresponding hypoxic marker intensities in adjacent sections. This permits vascular configuration to be related to oxygen availability and allows the hypoxic marker intensities to be quantitated in situ.

Microcirculatory model relating geometrical variation to changes in pressure and flow rate

A primary first step in defining the functional capabilities of a given microcirculatory bed is to describe the distribution of intravascular pressure and flow throughout the network using an idealized geometrical model. By applying an approach developed by the geomorphological sciences, changes in vessel lengths, numbers, and diameters were systematically related to order number by applying three mathematical relationships. From measurements of a number of vascular modules, generalized relationships were obtained which applied to the microcirculation as a whole. An idealized model was developed by the computer-generation of node-node connections based on their probabilities of occurrence, and by generation of lengths and diameters, based on equations relating diameter and length to branching order number. Networks of this type were readily analyzable in terms of pressure-flow characteristics, using linear network analysis techniques. The current study focused on two specific microvascular beds: [1] rabbit omentum, in which numerous studies have related in vivo pressures and flows to geometry, and [2] human bulbar conjunctiva, in which quantitative data is almost totally nonexistent. In view of the close correlation between measured and predicted omental pressure and flow values, it was apparent that the current modeling scheme offered a close approximation to in vivo values. A more significant application, however, is in the correlation of complex alterations in vascular geometry with the accompanying hydrodynamical changes.

Analysis of the effects of measured white blood cell entrance times on hemodynamics in a computer model of a microvascular bed

To quantify the interdependence of capillary leukocyte plugging and microvascular hemodynamics, experimental measurements were made of the time required for lymphocytes and granulocytes to enter a micropipette. Using standard micropipette deformation techniques, entrance times were found to be a function of both cell diameter and pipette diameter, with no significant dependence on aspiration pressure over the differential pressure range of 200–400 Pa. Experimental results were combined with a computer network model to describe changes in red cell distribution and flow rate resulting from the delayed entrance of leukocytes (WBC) into capillaries. The network model is based on geometrical measurements from the capillary bed of a hamster cremaster muscle (Sarelius et al. 1981) and utilizes previous work describing: 1. preferential cell distribution at a bifurcation, 2. increased apparent viscosity due to the presence of red and white cells, and 3. increased velocities of red and white cells relative to blood. Red and white cell positions within the network were computed at discrete time increments, and WBC plugging was simulated by a temporary cessation of flow in vessels of smaller diameter than the white cell. In contrast with previous studies, the increased viscosity due to the presence of WBCs was found to have an insignificant effect on overall network flow rate. Instead, a major flow reduction occurs only when capillaries are plugged by the white cells. At normal physiological concentrations (1,000 RBC/WBC), time-averaged overall network flow is reduced by 4.4%, based on averaged experimentally measured entrance times, and up to 14.8% if maximal entrance times are used. Overall flow rates fluctuate significantly with time and, at concentrations greater than 1 WBC/200 RBC, decrease substantially with increasing WBC concentration. At levels comparable to acute leukemia (70 RBC/WBC), flow is severely compromised due to numerous WBC occlusions. In the absence of WBCs, mean hematocrit and hematocrit distribution compare well with published values. Spatial variations in hematocrit are notable, however, and capillaries originating from the upstream end of the arteriole have a much lower hematocrit than downstream branches (by a factor of two, on average) due to preferential distribution of red blood cells.

Intracapillary HbO2 saturations in murine tumours and human tumour xenografts measured by cryospectrophotometry: Relationship to tumour volume, tumour ph and fraction of radiobiologically hypoxic cells

Frequency distributions for intracapillary HbO2 saturation were determined for two murine tumour lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI) using a cryospectrophotometric method. The aim was to search for possible relationships between HbO2 saturation status and tumour volume, tumour pH and fraction of radiobiologically hypoxic cells. Tumour pH was measured by 31P NMR spectroscopy. Hypoxic fractions were determined from cell survival curves for tumours irradiated in vivo and assayed in vitro. Tumours in the volume range 100-4000 mm3 were studied and the majority of the vessels were found to have HbO2 saturations below 10%. The volume-dependence of the HbO2 frequency distributions differed significantly among the four tumour lines; HbO2 saturation status decreased with increasing tumour volume for the KHT, RIF-1 and MLS lines and was independent of tumour volume for the OWI line. The data indicated that the rate of decrease in HbO2 saturation status during tumour growth was related to the rate of development of necrosis. The volume-dependence of tumour pH was very similar to that of the HbO2 saturation status for all tumour lines. Significant correlations were therefore found between HbO2 saturation status and tumour pH, both within tumour lines and across the four tumour lines, reflecting that the volume-dependence of both parameters probably was a compulsory consequence of reduced oxygen supply conditions during tumour growth. Hypoxic fraction increased during tumour growth for the KHT, RIF-1 and MLS lines and was volume-independent for the OWI line, suggesting a relationship between HbO2 saturation status and hypoxic fraction within tumour lines. However, there was no correlation between these two parameters across the four tumour lines, indicating that the hypoxic fraction of a tumour is not determined only by the oxygen supply conditions; other parameters may also be important, e.g. oxygen diffusivity, rate of oxygen consumption and cell survival time under hypoxic stress.

Alteration of tumour response to radiation by interleukin-2 gene transfer

We have previously shown that BALB/c-derived EMT6 mammary tumours transfected with interleukin (IL)-2 have decreased hypoxia compared to parental tumours, due to increased vascularization. Since hypoxia is a critical factor in the response of tumours to radiation treatment, we compared the radiation response of IL-2-transfected tumours to that of parental EMT6 tumours. Because the IL-2 tumours have an altered host cell composition, which could affect the interpretation of radiation sensitivity as measured by clonogenic cells, we employed flow cytometric analysis to determine the proportion of tumour cells vs host cells in each tumour type. Using this approach, we were able to correct the plating efficiency based on the number of actual tumour cells derived from tumours, making the comparison of the two types of tumours possible. We also excluded the possibility that cytotoxic T-cells present in EMT6/IL-2 tumours could influence the outcome of the clonogenic cell survival assay, by demonstrating that the plating efficiency of cells derived from EMT6/IL-2 tumours remained unchanged after depletion of Thy-1+cells. The in vivo radiation response results demonstrated that IL-2-transfected tumours were more sensitive to radiation than parental EMT6 tumours. The hypoxic fraction of the EMT6/IL-2 tumours growing in vivo was markedly decreased relative to parental EMT6 tumours thus the increased sensitivity results from the increased vascularity we have previously observed in these tumours. These results indicate the potential therapeutic benefit of combining radiation and immunotherapy in the treatment of tumours.

Combination of radiation and celebrex (celecoxib) reduce mammary and lung tumor growth.

The selective cyclooxygenase (COX)-2 inhibitor, celecoxib, alone and in combination with radiation was investigated in vitro and in vivo. Murine mammary tumor line (MCa-35) and human lung carcinoma line (A549) have high and low basal levels of COX-2 protein, respectively. Treatment of both tumor cells with celecoxib alone resulted in a dose- and time-dependent reduction of cell number (clonogenic cell death) and tumor cell growth rate in vitro; however, inhibition of tumor cell growth by celecoxib was not correlated with the reduction of COX-2 protein in tumor cells. Although both tumor cell types had similar DNA damage after celecoxib treatment, significant induction of tumor cell apoptosis was only observed in MCa-35. Celecoxib-mediated radiation sensitization also occurred in MCa-35 cells determined by clonogenic assay, in part due to a G2/M arrest at 8 to 24 hours after treatment. The tumor growth inhibitory effects of celecoxib were also studied in vivo. It was found that celecoxib inhibited both tumor growth after intragastric administration of celecoxib (5 daily doses of 50 mg/kg). Combined with a single 30-Gy dose of radiation, celecoxib resulted in additive effects on A549 tumors. Celecoxib-treated A549 tumors had marginal reduction of total and perfused blood vessels compared with untreated controls. Reduction of tumor angiogenic cytokine and growth factor mRNA was associated with decreased perfused vessels. Finally, reduction of vascular endothelial growth factor protein after celecoxib was also observed in both tumor lines by Western blot. Our results indicate that the selective inhibition of COX-2 combined with radiation has potential application in radiotherapy, and celecoxib-mediated antitumor effects may act through different mechanisms including direct inhibition of tumor cell proliferation, alteration of tumor cell cycle, and antiangiogenesis.

Keratinocyte growth factors radioprotect bowel and bone marrow but not KHT sarcoma

Various members of the fibroblast growth factor (FGF) family of proteins have been shown to protect against acute and late radiation damage of normal tissues. Protection of the small bowel, for example, occurs via both increased proliferation and reduced apoptosis. Other beneficial effects of FGFs include promotion of bone growth, pneumonitis prevention, and apoptosis suppression of endothelium in vivo and in vitro after irradiation. This protection against radiation requires only low and infrequent doses of FGFs. Two newly identified members of the FGF family, FGF7 and FGF10, have effects similar to many of the other FGF family proteins, but with more specificity for normal epithelial structures. For this reason, they have also been named keratinocyte growth factors one and two (KGF1 and KGF2, respectively). We therefore examined the potential utility of KGFs for radioprotection of the bone marrow and small bowel and examined safety issues concerning their adverse effects on KHT sarcoma. The results suggest that KGFs could be safely used to prevent radiation toxicity of the abdomen or pelvis and may in fact improve tumor response to radiation.

Enhanced Antitumor Effect of Combined Triptolide and Ionizing Radiation

Purpose: The lack of effective treatment for pancreatic cancer results in a very low survival rate. This study explores the enhancement of the therapeutic effect on human pancreatic cancer via the combination of triptolide and ionizing radiation (IR). Experimental Design:In vitro AsPC-1 human pancreatic cancer cells were treated with triptolide alone, IR alone, or triptolide plus IR. Cell proliferation was analyzed with sulforhodamine B (SRB) method and clonogenic survival; comparison of apoptosis induced by the above treatment was analyzed by annexin V–propidium iodide (PI) staining. Furthermore, the expression of apoptotic pathway intermediates was measured by the assay of caspase activity and Western blot. Mitochondrial transmembrane potential was determined by JC-1 assay. In vivo, AsPC-1 xenografts were treated with 0.25 mg/kg triptolide, 10 Gy IR, or triptolide plus IR. The tumors were measured for volume and weight at the end of the experiment. Tumor tissues were tested for terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) and immunohistochemistry. Results: The combination of triptolide plus IR reduced cell survival to 21% and enhanced apoptosis, compared with single treatment. In vivo, tumor growth of AsPC-1 xenografts was reduced further in the group treated with triptolide plus IR compared with single treatment. TUNEL and immunohistochemistry of caspase-3 cleavage in tumor tissues indicated that the combination of triptolide plus IR resulted in significantly enhanced apoptosis compared with single treatments. Conclusions: Triptolide in combination with ionizing radiation produced synergistic antitumor effects on pancreatic cancer both in vitro and in vivo and seems promising in the combined modality therapy of pancreatic cancer.