Loss of myocardial capillary endothelial-cell alkaline phosphatase (ALP) activity in primary endothelial cell culture.

Primary cultures of rat myocardial capillary endothelial cells were established and characterized. A range of typical endothelial cell-specific markers were retained in vitro. Cell kinetic studies in confluent endothelial-cell cultures in vitro revealed a roughly 50-fold increase in the proportion of cells in s-phase, indicating a very considerable shortening of cell turnover time, compared to in vivo conditions. Alkaline phosphatase enzyme activity and encoding mRNA are strongly expressed in myocardial capillary endothelial cells in vivo, but were not detectable in vitro. This was true in cell cultures from two strains of rat, which revealed significantly different enzyme expression levels in vivo. In co-cultures of pericytes and endothelial cells, positive ALP enzyme reaction was detected in pericytes, which in vivo show only very weak enzyme reactivity. Treatment of cell cultures with

Radiation-induced impairment of bone healing in the rat femur: effects of radiation dose, sequence and interval between surgery and irradiation.

BACKGROUND AND PURPOSE: Impairment of osseous healing in treatment combining surgery and radiotherapy is a frequent complication. Its dependence on sequence and interval was studied in a defined experimental model. MATERIALS AND METHODS: The effect of pre- and postoperative irradiation by single doses of X-rays on osseous closure of a 1.2 mm drill hole in the rat femur was measured 6 or 7 weeks after surgery in histological sections using morphometrical methods. RESULTS: Irradiation delivered between 1 day and 6 months before surgery resulted in a reduction of bone healing following very similar dose response relationships; there was no evidence of any slow repair of latent radiation damage. Radiosensitivity of bone healing during the first 3 days after surgery was not different from preoperative irradiation; however, irradiation 4 days or later after surgery failed to reduce osseous healing even after very high radiation doses. CONCLUSION: Tolerance increases enormously if radiotherapy is given later than 4 days after surgery. This has great implications for combined radiotherapy and surgery schedules involving bone reconstruction, but may be even more important for radiotherapy applied to prevent heterotopic ossification after total hip arthroplasty. Biologically, target cell regeneration alone is insufficient to account for the drastic rise in radiotolerance; it must be accompanied by an increase in cellular resistance due to differentiation.

Peripheral blood mononuclear cells induce programmed cell death in human endothelial cells and may prevent repair: role of cytokines.

Human umbilical vein endothelial cells (HUVECs) undergo programmed cell death (apoptosis) after coculture with peripheral blood mononuclear cells (PBMCs) preactivated by ionizing radiation (IR) or by bacterial endotoxin (lipopolysaccharide [LPS]). Cell-to-cell contact-mediated apoptosis could be blocked in both cases by anti-tumor necrosis factor-alpha (anti-TNF-alpha) monoclonal antibody MAK195 and also by the antagonistic cytokine interleukin-10 (IL-10). Cell-free PBMC supernatants from both preactivation treatments were sufficient to trigger endothelial apoptosis. In contrast, MAK195 and IL-10 were found to be ineffective in this system, suggesting a TNF-alpha-independent mechanism. However, N-Acetylcystein, an antioxidant, fully abrogated programmed cell death mediated by the supernatant of IR-treated PBMCs, but not of LPS-treated PBMCs. Additionally, we found that coculture and cell-free supernatants of preactivated as well as untreated PBMCs caused cell cycle arrest in proliferating EC in G(0/1), which could be relieved by IL-10, but not by anti-TNF-alpha. Further analysis showed that transforming growth factor-beta, which was constitutively expressed in the supernatant of PBMCs, namely lymphocytes, was responsible for this. These data suggest a pathophysiologic model in which preactivated PBMCs cause EC damage and may prevent blood vessel repair by arresting the proliferation of ECs. This could contribute to the understanding of various clinical endothelial complications that occur after irradiation as well as in cases of endotoxemia or related inflammatory states.

Radiation-induced changes in transforming growth factor beta and collagen expression in the murine bladder wall and its correlation with bladder function.

Late radiation-induced changes in transforming growth factor beta (TGF-beta), collagen I and collagen III content of the bladder wall, as well as morphological alterations of the uroepithelium, were analyzed quantitatively in an immunohistochemical study. An interlaboratory, i.e. interstrain, comparison of two mouse strains (Amsterdam C3H/Hen Af-nu+ and Munich C3H Neu) with different dose-effect relationships for late bladder damage was made, choosing radiation doses producing equivalent functional alterations in both strains (ED80 of 25 Gy and 19 Gy, respectively, 40 weeks after irradiation). In one strain of mouse, cystometry was also performed in the same animals at different times after irradiation. The TGF-beta staining intensity showed a progressive increase between 90 and 360 days after irradiation. This increase was similar in both strains of mouse treated with functionally equivalent doses (ED80) and was less pronounced after a lower, ED40, dose in the Munich mice. In both strains, there was a radiation-induced increase in both collagen subtypes from 180 days after irradiation with the ED80. The ratio of collagen type I/III, however, decreased in the Amsterdam mice and increased in the Munich mice. The relative radioresistance of the Amsterdam mice may therefore be partly due to a greater contribution of the elastic collagen type III, affording greater bladder compliance after irradiation. The extent of radiation-induced uroepithelial denudations or papillomatous outgrowths, the TGF-beta staining intensity and collagen I/III ratio were each correlated to bladder function determined by cystometry for the Munich mice. This correlation was statistically significant for all three parameters for group mean responses and, with the exception of the collagen I/III ratio, also for individual mice. These experiments indicate that chronic radiation-induced alterations in TGF-beta expression and connective tissue metabolism in the bladder wall are possibly important factors determining reduced bladder function after irradiation.

Early inflammatory reaction of the rabbit coeliac artery wall after combined intraoperative (IORT) and external (ERT) irradiation.

The present immunohistochemical study of radiation-induced damage in major blood vessels is based on a multidisciplinary study (Schultz-Hector et al., Radiother. Oncol., 38: 205-214, 1996) investigating the combined effect of IORT of the coeliac axis and upper abdominal ERT. The paper describes the sequential changes occurring in the coeliac artery after IORT with 30 Gy, i.e. during and after combined IORT and fractionated ERT (total dose 40 Gy). Within 24 h after IORT, the arterial wall was found to be invaded by TNF-alpha positive macrophages, which later on disappeared within 7-14 days. At 2 days post-IORT, the medical smooth muscle cells were strongly positive for TNF-alpha and remained positive throughout the observation period of 63 days. At 80 days, a comparison of different IORT dose groups showed that TNF-alpha expression after 20 and 30 Gy IORT plus 40 Gy ERT had subsided, while it was still strongly evident after 40 Gy IORT. Negative reactions in sham irradiated animals or animals treated with ERT alone indicate that TNF-alpha expression was caused by IORT. After > 30 days post-IORT, there was increased collagen type I deposition in the adventitia. In two animals receiving the full ERT course, intimal proliferations involving mainly smooth muscle cells were observed. Our findings indicate that some features typical of radiation induced arteriosclerosis such as periarterial fibrosis and intimal proliferations can occur as early as < 60 days postirradiation. Macrophage invasion as well as TNF-alpha expression in medial smooth muscle cells are known to be important steps in the development of spontaneous atherosclerotic lesions. Therefore, early TNF-alpha induction in the arterial wall by a high local dose of X-irradiation may be regarded as one initiating factor of chronic radiation-induced arteriosclerosis.

Complications of combined intraoperative radiation (IORT) and external radiation (ERT) of the upper abdomen: an experimental model.

An experimental model in the rabbit is presented which is suitable for analysis of clinically relevant, early side-effects of combined upper abdominal IORT and ERT. Fractionated ERT alone given through an upper abdominal a.-p. field including the entire stomach caused gastric ulcerations within < or = 58 days. Latent times decreased with increasing dose and the ED50 for occurrence of ulcers was 39 +/- 3.3 Gy. Single doses of IORT of 20-40 Gy alone administered through a 2-cm diameter field localized on the coeliac axis and carefully excluding any intestinal mucosa caused neither gastric ulcerations nor other clinical symptoms. When ERT with 40 Gy was preceded by IORT with 20-40 Gy or by sham IORT, 13 out of 15 animals developed ulcers after latent times which in a life-table analysis were shown to be significantly shorter than after ERT alone. However, a statistically significant IORT dose-dependence of latent time or incidence of ulcers could not be demonstrated in the present experiment. The most significant histological changes were observed in the areas of gastric ulcers. Already during ERT, the mucosal epithelium was depleted and regenerative activity was evident in spite of ongoing fractionated irradiation. However, profound irregularities in glandular structure and distribution, as well as number of proliferating epithelial cells were still present in healed ulcers at 80 days. In summary, IORT to the coeliac artery did precipitate the development of gastric ulcers induced by subsequent ERT. On the one hand, the data indicate that the surgical procedure of IORT did contribute to this effect. On the other hand, IORT to the coeliac artery could cause transient, functional alterations in blood supply to the depending organs, i.e. the stomach, and could thus precipitate the development of radiation-induced ulcers.

[Radiation sequelae in the large arteries. A review of clinical and experimental data]. / Strahlenfolgen an grossen Arterien. Ubersicht über klinische und experimentelle Daten.

BACKGROUND: In the course of radiation therapy of malignant tumors, inclusion of major arteries into the radiation field is often inevitable. PATIENTS AND METHODS: Clinical case reports and studies were compiled and analyzed with respect to the effect of irradiation on the risk of arteriosclerotic changes within the radiation field. The study concentrates on those anatomic locations which are involved frequently enough in order to allow a quantitative analysis. RESULTS: In a series of clinical studies, a consistent 3- to 4-fold increase in carotis stenoses is observed following radiation therapy of head and neck tumors. The majority of clinically symptomatic stenoses, however, is not observed earlier than 8 years post irradiation. Although observations in other peripheral arteries do not allow to estimate incidences, they do confirm, however, the finding of a very long latent time. Following mediastinal or thoracic wall irradiation, the risk of coronary artery disease is significantly increased after follow-up times of > or = 10 years. Radiation related arterial injury is sharply limited to arterial segments included in the treatment field and is often observed in unusual locations. The histological appearance and development however, is not fundamentally different from lesions observed in cases of generalized arteriosclerosis. Experimental observations indicate that patients with general arteriosclerosis risk factors might have a particularly increased risk of developing arterial injury following therapeutic irradiation. CONCLUSION: Irradiation of large blood vessels in the course of tumor therapy represents a long-term local risk factor for development of arteriosclerosis.

Quantitative distinction of cisplatin-sensitive and -resistant mouse fibrosarcoma cells grown in multicell tumor spheroids.

As a suitable model to study the growth behavior and therapeutic response of drug-resistant and -sensitive cells in three-dimensional coculture we have established multicellular spheroids generated from both cisplatin-sensitive and -resistant cells of a murine fibrosarcoma cell line. A drug resistant clone was derived from the parent cisplatin-sensitive cells by intermittent drug exposure in vitro. As a prerequisite for analysis of differential growth and treatment response of spheroid subpopulations, two efficient methods to discriminate between the two morphologically indistinguishable subpopulations in mixed spheroids were established. In the cisplatin-resistant cell line chosen for the present study, resistance is mainly due to an increased cellular metallothionein content and is therefore associated with increased resistance to CdCl2. Exposure of colonies to high concentrations of CdCl2 thus allowed selective elimination of sensitive colonies. Permanent labeling of either resistant or sensitive cells was achieved by introduction of the Escherichia coli beta-galactosidase marker gene with a retroviral vector system. The transformation of an uncolored galactose derivative by this enzyme into an indigo stain allowed detection of cells carrying and expressing the marker gene. The marker gene and CdCl2 method led to identical results when used simultaneously to distinguish quantitatively between resistant and sensitive colonies grown from plated cells of untreated or irradiated mixed spheroids. The retroviral labeling method was also used successfully in the study of intact spheroids, showing that in 1:1 mixed spheroids, cisplatin-sensitive parent cells accumulate in the spheroid periphery, outgrowing resistant cells and displacing them into the metabolically restricted spheroid center. Only when sensitive and resistant cells are initially mixed at a ratio of 1:9 are the resulting spheroids composed of equal proportions of the 2 cell types throughout 10-20 days after spheroid initiation.

Radiation-induced loss of endothelial alkaline phosphatase activity and development of myocardial degeneration. An ultrastructural study.

BACKGROUND: There is general agreement that radiation effects on capillary endothelial cells are a leading event in the pathogenesis of late effects of radiation in normal tissues. The mechanism of microvascular involvement however is unclear. In the myocardium, there is not only a decrease in capillary number, but a focal loss of endothelial alkaline phosphatase. The present study addresses the question of whether radiation-induced alkaline phosphatase loss is due to cell death or to modification of cell function and ultrastructure. EXPERIMENTAL DESIGN: The time course of ultrastructural changes underlying endothelial alkaline phosphatase loss and development of myocardial degeneration was studied in two strains of rat, that differ in latent time of clinical radiation-induced cardiomyopathy. RESULTS: In both strains of rat, development of ultrastructural damage in cardiomyocytes was preceded by a focal loss of endothelial alkaline phosphatase reactivity. The absence of enzyme reaction product was neither due to endothelial cell loss, nor to a depletion in enzyme-bearing cytotoxic vesicles. The endothelial cell/pericyte relationship was also unchanged. Within enzyme-negative areas, there was an increased number of enlarged endothelial cells and of lymphocyte adherence to endothelial cells, which was then followed by endothelial cell rupture and extravasation of blood cells. In Wistar rats, enzyme loss started at 25 days after 20 Gy and reached its maximum extent by 90 days. In Sprague-Dawley rats, which show a significantly higher pre-irradiation enzyme reactivity, the onset of alkaline phosphatase loss and associated alterations was delayed by about 30 days and was significantly less extensive. CONCLUSIONS: Radiation-induced endothelial alkaline phosphatase loss is unrelated to cell death in mitosis, but nonetheless it is relevant for the development of ultimate clinical heart failure.

Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques.

A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([3H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase anti-alkaline-phosphatase technique, the peroxidase-anti-peroxidase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a G0/G1 peak nor a G2 + M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.

Cellular localization of endothelial alkaline phosphatase reaction product and enzyme protein in the myocardium.

Myocardial capillary endothelial cells, arteriolar endothelial cells, and the arterial adventitia show positive alkaline phosphatase (AP) enzyme reaction and immunoreactivity in both rat and human hearts. In guinea pigs, however, capillary endothelial staining is discontinuous and arterial adventitia is negative. The ultrastructural correlate of discontinuous capillary staining is a pronounced labeling of pericytes in guinea pig heart and relatively weak endothelial staining. In rat and human heart, enzyme reaction products are localized mainly on plasma membranes and cytotic vesicles of endothelial cells. Comparison of two strains of rat reveals a more dense deposition of enzyme reaction product along the luminal and particularly along the abluminal plasma membrane of Sprague-Dawley rats than of Wistar rats. Quantitative analysis of immunogold labeled anti-AP antibody density confirms the pronounced polarity of capillary endothelial cell labeling in Sprague-Dawley rats. More than 80% of total endothelial AP protein in Sprague-Dawley rats is localized over the abluminal plasma membrane and basal lamina, as compared with less than 30% in Wistar rats. Moreover, the total endothelial cell labeling is almost sixfold higher in Sprague-Dawley than in Wistar rats. Total endothelial labeling and proportion of labeling on the abluminal endothelial plasma membrane in human hearts is intermediate between the two strains of rat. The strain and species differences in enzyme distribution could provide important information concerning enzyme function.

Cell kinetic analysis of murine squamous cell carcinomas: a comparison of single versus double labelling using flow cytometry and immunohistochemistry.

The study was originally set up to measure accurate cell kinetic parameters in two murine squamous cell carcinomas (scc) for comparison with radiobiological data on proliferation during radiotherapy. The tumours, AT84 and AT478, were both moderately well differentiated aneuploid scc. In the course of the study, several comparisons of techniques were made in two different centres. This paper reports on the results of those comparisons involving two different detection methods (flow cytometry and immunohistochemistry), single vs double labelling, and in vivo and in vitro labelling, the latter using tissue slices incubated under high pressure oxygen. Pulse labelling studies with bromodeoxyuridine (BrdUrd) showed that the labelling indices (LI) were not significantly different after in vitro or in vivo labelling. In addition, the flow cytometry (FCM) and immunohistochemistry (IHC) methods also gave labelling indices which were not significantly different. Only tumour cells were analysed in these studies by selecting cells on the basis of aneuploidy (FCM) or morphology (IHC). The DNA synthesis time of the tumour cells were analysed by both techniques. For FCM, the Relative Movement method was used (Begg et al., 1985). For IHC, a double labelling method was used, employing BrdUrd and triated thymidine (3H-TdR) administered several hours apart, detected simultaneously using immunoperoxidase and autoradiography, respectively. When both labels were administered in vivo, there was good agreement for Ts between the FCM and IHC methods. Attempts were also made to measure Ts in vitro using both techniques. With double labelling, it was found that cells did not take up the second label, implying a failure of cycle progression. This was confirmed by FCM results, showing no movement of labelled cells through the S-phase, despite an initially high uptake. This could not be influenced by lowering the DNA precursor concentration or by adding foetal calf serum. This indicates that DNA synthesis times are difficult or impossible to measure in vitro in fresh tumour explants. Finally, the double labelling IHC method allowed intratumoural variations of both LI and Ts to be studied. Both parameters were found to vary markedly throughout the tumour volume, particularly for larger tumours (600 mg), giving calculated local potential doubling time values (Tpot) ranging from 1-7 days.

Beta-fibroblast growth factor expression in human and murine squamous cell carcinomas and its relationship to regional endothelial cell proliferation.

Surgical biopsies from ten head and neck squamous cell carcinomas were labeled in vitro with bromodeoxyuridine. In histological sections, bromodeoxyuridine-positive nuclei and beta-fibroblast growth factor (beta-FGF) were stained using immunohistochemistry. In clearly discernible clusters of tumor cells, the cytoplasm shows strong positive beta-FGF staining, whereas other tumor regions are completely beta-FGF negative. Within positively stained areas, the tumor cell bromodeoxyuridine labeling index is higher in comparison to beta-FGF-negative areas by a factor of 5 +/- 0.8. This is reflected in a positive correlation of the tumor cell labeling index and the relative extent of beta-FGF-positive tumor areas. Viable tumor areas bordering on necrosis, which are known to be hypoxic, are beta-FGF negative. The average tumor endothelial cell labeling index was 1.8 +/- 0.6%, as compared to 0.16% in adjacent normal mucosa. Since endothelial cell pulse labeling indices are too low for a further quantitative analysis, the relationship of beta-FGF expression and endothelial cell turnover was studied in more detail in two fairly well-differentiated murine squamous cell carcinoma lines (AT 84 and AT 478). Labeling indices were higher and endothelial cell doubling times were significantly shorter in beta-FGF-positive as compared to beta-FGF-negative tumor areas (AT 84, 9.3 h versus 25.4 h; AT 478/25, 6.8 h versus 16 h). Thus, the discrete expression of beta-FGF is associated with regional differences in endothelial cell kinetics. In two generations of the tumor line AT 478, characterized by different volume doubling times of 18 days (AT 478/25) and 36 days (AT 478/4), beta-FGF-positive areas represent 75.5 +/- 6% and 19.7 +/- 7% of the viable tumor tissue, respectively. This indicates a correlation between beta-FGF production of tumor cells and growth rate.

Early changes in mouse urinary bladder function following fractionated X irradiation.

Functional changes in the mouse urinary bladder following single-dose or fractionated irradiation were assessed by cystometry, i.e., by measuring the intravesical volume-pressure relationship during transurethral filling. The early response presented as a dose-dependent and transitory decrease in the reservoir function of the organ as defined by the intravesical volume at a filling pressure of 10 or 20 mm Hg, V10 or V20. The quantal dose response used in the present study was a reduction in the individual bladder volume (V10 and V20) by at least 50%. After single doses greater than or equal to 10 Gy, the reaction occurred between Days 7 and 25 with maximum prevalence between Days 7 and 14. The individual duration of the response was 3-9 days. Treatment with single doses and 2, 3, 5, and 10 fractions demonstrated a significant sparing effect with ED50 values of 18.3, 24.9, 26.8, 29.8, and 38.0 Gy, respectively. The linear-quadratic model fitted the data reasonably well when tested according to Tucker (Int. J. Radiat. Oncol. Biol. Phys. 10, 1933-1939, 1984). The alpha/beta ratios estimated with different two-step techniques ranged from 11.1 to 12.4 Gy. Analysis of the data as proposed by Thames et al. (Int. J. Radiat. Biol. 49, 999-1009, 1986) yielded an alpha/beta value of 13.9 Gy (95% confidence limits 8.4 and 24.6 Gy), illustrating a fractionation effect typical for acutely responding tissues, although no clear cell depletion occurred in the urothelium.

Radiation-induced heart disease: morphology, changes in catecholamine synthesis and content, beta-adrenoceptor density, and hemodynamic function in an experimental model.

To study further the pathophysiology of radiation-induced cardiomyopathy, we investigated resting hemodynamics, myocardial catecholamine synthesis and storage, and beta-adrenoceptor density after local heart irradiation. In Wistar rats, a radiation dose of 20 Gy eventually leads to compromised myocardial function which is characterized by a reduction in cardiac output to 43 +/- 11% and in the left ventricular ejection fraction to 66 +/- 7.5%, and an increase in the left ventricular end-diastolic volume to 187 +/- 17% of control values. This reduction in function is correlated with focal degeneration of 23 +/- 4% of the myocardium. Measurement of tyrosine hydroxylase activity and catecholamine content revealed that catecholamine biosynthesis is unchanged in the adrenals but is significantly reduced in the hearts of irradiated animals, while cardiac beta-adrenoceptor density is increased to about 140% of that in age-matched controls. This is in contrast to findings in dilated or ischemic cardiomyopathy. Time-course studies showed that the development of myocardial degeneration starts simultaneously with the decrease in cardiac output and ejection fraction and the increase in beta-adrenoceptors at 50-80 days postirradiation. Myocardial degeneration is maximal in extent and severity at 100 days and does not progress thereafter. Cardiac output decreases at 80-100 days postirradiation to 60 +/- 7% of control values. A significant further decrease is seen only when congestive heart failure becomes manifest at 249 +/- 21 days after 20 Gy. Thus there is a delay between structural myocardial injury and hemodynamic deterioration which could be due to a compensatory increase in beta-adrenoceptor density during the initial stages of the cardiomyopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiation-induced heart disease: review of experimental data on dose response and pathogenesis.

Clinical and experimental heart irradiation can cause a variety of sequelae. A single dose of greater than or equal to 15 Gy leads to a reversible exudative pericarditis, occurring in dogs, rabbits or rats at around 100 days. Its time-course is very similar in all species investigated, but there are considerable species and strain differences in severity and incidence. After longer, dose-dependent latency times chronic congestive myocardial failure develops. At histological examination myocardial degeneration and necrosis is observed, which in some species is accompanied by a variable degree of interstitial fibrosis. In rabbits and rats, myocardial degeneration becomes apparent at around 70 days after 20 Gy and is preceded by a marked reduction in capillary density as well as ultrastructural endothelial cell degeneration. Simultaneously to structural capillary damage, a focal loss of the endothelial marker enzyme alkaline phosphatase was observed in rats in areas with subsequent myocardial degeneration. Cell kinetic studies in rabbits and rats revealed a radiation-induced wave of increased endothelial cell proliferation at 30-100 days postirradiation. In the rat it is exclusively seen in conjunction with alteration of endothelial cell marker enzymes. The temporal and spatial pattern of proliferative response exludes endothelial cell death in mitosis as the sole pathogenetic mechanism causing capillary loss and myocardial degeneration. Parallel to development of morphological damage, haemodynamic studies in various rats strains revealed a drop in cardiac output and left ventricular ejection fraction to about 64% of normal values after 20 Gy. In vivo, this slightly reduced cardiac function was then maintained in a steady state for many weeks, probably due to a compensatory up-regulation of cardiac beta-adrenergic receptors. In denervated working heart preparations in vitro, however, these compensatory mechanisms are not effective and stroke volume as well as cardiac contractility show a rapid and steady deterioration. In many respects radiation-induced heart disease conforms to radiobiological concepts of late-responding tissues, showing a chronic progressive time-course and a very pronounced fractionation effect. However, pathogenesis cannot be understood in terms of target cell depletion alone, and experimental evidence indicates the importance of alterations of regulatory mechanisms.

Fractionation response and repair kinetics of radiation-induced heart failure in the rat.

Local heart irradiation with single or fractionated doses leads to heart failure after dose-dependent latency times. Clinical symptoms of heart failure are dyspnoea at rest, apathy and subcutaneous oedema. Animals autopsied when they presented with these symptoms, have a congested liver and occasional pleural effusions. The left ventricle is dilated, showing a reduction in wall thickness by 15-17% of control values. Histological examination reveals a focal degeneration and necrosis of about 23% of the total myocardial volume. Loss of alkaline phosphatase activity from myocardial capillaries, which is known to precede myocardial degeneration, involves 77% of the myocardium. These findings at the time of manifest heart failure are constant, independent on whether injury to the heart was inflicted by single-dose or fractionated irradiation or whether heart failure developed within a relatively short time after high total doses or within many months after low total doses. The latent time of heart failure therefore can be considered an appropriate endpoint for comparison of treatment groups. From experiments giving 1, 2, 4, or 10 dose fractions, a low alpha/beta ratio of 3.7 Gy (95% confidence interval 1.8-5.6 Gy) can be calculated. When the time interval between dose fractions is varied in a split-dose experiment, time intervals of up to 3 h do not increase the survival time significantly. This appears to indicate very slow repair of sublethal damage. On the other hand, it cannot be excluded that pathogenetic mechanisms independent of cell death in the renewing cell population contribute to this effect, making an interpretation of the alpha/beta ratio in terms of cell survival parameters of a defined target cell population difficult.