Long-term effects of intracoronary beta-radiation in balloon- and stent-injured porcine coronary arteries.

BACKGROUND: The data on the long-term safety and efficacy of intracoronary beta-radiation in animal models are limited. METHODS AND RESULTS: A total of 30 coronary arteries in 15 swine were subjected to balloon or stent injury followed by beta-radiation from a centered 32P source (2000 cGy to 1 mm beyond lumen surface) or a sham radiation procedure. The animals received aspirin for 6 months and ticlopidine for 30 days. Five of the 10 animals subjected to radiation died (at 5 days, 7 days, 3 months [n = 2], and 4 months) as a result of layered, occlusive thrombus at the intervention site (3 stent and 2 balloon injury sites). No deaths occurred in the control group. In the surviving animals, balloon-injured and irradiated vessels showed a trend toward larger lumens than controls (2.15 +/- 0.17 versus 1.80 +/- 0.08 mm2, P=0.06) and larger external elastic lamina areas (3.32 +/- 0.21 versus 2.62 +/- 0.10 mm2, P=0.003). In the stent-injured vessels from surviving animals, lumen, neointimal, and external elastic lamina areas were 3.58 +/- 0.33, 3.16 +/- 0.35, and 8.12 +/- 0.42 mm2 for irradiated vessel segments; these values were not different from those in controls (3.21 +/- 0.15, 2.84 +/- 0.27, and 7.76 +/- 0.28 mm2, respectively). Histologically, healing was complete in most survivors, although intramural fibrin and hemorrhage were occasionally seen. CONCLUSION: In the long-term (6 month) porcine model of restenosis, the inhibition by intracoronary beta-radiotherapy of the neointimal formation that is known to be present at 1 month is not sustained. This lack of effect on neointimal formation after balloon and stent arterial injury is accompanied by subacute and late thrombosis that leads to cardiac death on a background of continuous aspirin but relatively brief ticlopidine treatment.

Disc angiogenesis assay.

The aim of our research was to develop a quantitative assay for angiogenesis in mammals, especially the mouse. This is a convenient experimental animal because of its small size, which allows compact housing and experimentation with angiogenic factors or inhibitors in limited supply. Mouse genetics is an advanced discipline, resulting in the availability of many inbred strains and histocompatible tumors. Recombinant growth factors and other proteins are usually of human or mouse origin, and the desirability of using proteins of the experimental animal under study has been demonstrated. Mice genetically engineered to overproduce or not produce particular growth factors or receptors are valuable experimental tools.

Dose-response study of intracoronary beta-radiation with 32P in balloon- and stent-injured coronary arteries in swine.

PURPOSE: A dose-response study was performed in swine to investigate the vascular effects of 32P over a broad range of doses in order to define the therapeutic window of intracoronary radiotherapy (ICR) with 32P. METHODS AND MATERIALS: A total of 131 porcine arteries were subjected to balloon injury or stenting followed by 0-36 Gy of ICR from a centered 32P source wire to 1 mm beyond lumen surface or a sham ICR procedure. Animals were euthanized at 4 weeks, and vessels were harvested for histomorphometry. RESULTS: In the balloon-injured arteries, doses of 7 and 9 Gy did not impact restenosis. At doses of 14-36 Gy, neointima was markedly reduced, with mild dilatation at the highest dose, 36 Gy. In the stent-injured arteries, the lowest dose of 9 Gy failed to reduce neointimal growth, while 14-26 Gy showed the most favorable response. CONCLUSIONS: ICR with 32P features a broad therapeutic window. Doses of 14-26 Gy to 1 mm beyond lumen surface provided an optimal combination of efficacy and safety. Doses of 7 and 9 Gy were generally ineffective, suggesting a minimum threshold for ICR with 32P to effectively inhibit restenosis.

Acute radiation proctitis: a sequential clinicopathologic study during pelvic radiotherapy.

PURPOSE: Rectal toxicity is often dose limiting during pelvic radiation therapy. This prospective study examined the sequential development and associations of clinical, endoscopic, and histopathologic rectal toxicity during ongoing radiation therapy. METHODS AND MATERIALS: Thirty-three patients with nongastrointestinal pelvic carcinomas underwent proctoscopy with biopsy before radiation therapy, after 2 weeks treatment, and toward the end of the treatment course (6 weeks). Symptoms of acute toxicity were recorded, and endoscopic changes were graded. Histologic changes in the surface epithelium, glandular layer, and lamina propria were assessed using an ad hoc scoring system. Macrophage accumulation was evaluated in anti-CD68 stained sections. RESULTS: Pretreatment endoscopy and biopsies were unremarkable. Clinical symptoms progressed toward the end of the treatment course. In contrast, endoscopic pathology was maximal at 2 weeks. Biopsies obtained during treatment exhibited atrophy of the surface epithelium, acute cryptitis, crypt abscesses, crypt distortion and atrophy, and stromal inflammation. Histologic changes, particularly those in the surface epithelium, were consistently more pronounced at 2 weeks than they were at 6 weeks. CONCLUSION: In contrast to clinical symptoms, endoscopic changes stabilize and histologic changes regress from the 2nd to the 6th week of treatment. These results may have implications for the design and timing of prophylactic and therapeutic interventions to reduce radiation proctitis.

Angiogenesis is impaired by hypercholesterolemia: role of asymmetric dimethylarginine.

BACKGROUND: Many angiogenic factors require endothelium-derived nitric oxide (NO) to exert their effects. Recently, an endogenous competitive antagonist of NO synthase has been characterized: asymmetric dimethylarginine (ADMA). Elevated plasma levels of ADMA reduce NO synthesis in hypercholesterolemia. Accordingly, we hypothesized that hypercholesterolemia impairs angiogenesis by an ADMA-dependent mechanism. METHODS AND RESULTS: Angiogenesis was assessed with the use of a disk angiogenesis system implanted subcutaneously in normal (E(+)) mice or apolipoprotein (apo)E-deficient hypercholesterolemic (E(-)) mice. After 2 weeks, the disks were removed, and the fibrovascular growth area was used as an index of angiogenesis. Basal and fibroblast growth factor-stimulated angiogenesis was impaired in E(-) mice, associated with an elevation in plasma ADMA. Oral administration of L-arginine reversed the impairment of angiogenesis in E(-) mice. By contrast, oral administration of L-nitroarginine (an exogenous antagonist of NO synthase) reduced angiogenesis. When added directly to the disk, ADMA dose-dependently inhibited basal and fibroblast growth factor-induced angiogenesis, an effect that was reversed by oral administration of L-arginine. CONCLUSIONS: The derangement of the NO synthase pathway that occurs in hypercholesterolemia is associated with an impairment of angiogenesis. The lipid-induced impairment of angiogenesis can be reversed by oral administration of L-arginine and can be mimicked in normocholesterolemic animals by administration of an NO synthase antagonist. The data are consistent with the hypothesis that ADMA is an endogenous inhibitor of angiogenesis.

Despite its homology to angiostatin apolipoprotein(a) does not affect angiogenesis.

Apolipoprotein(a) [apo(a)] contains a kringle domain(IV) homologous to that of angiostatin, a natural angiogenic inhibitor. Because of this structural similarity we suspected that apo(a) could be an inhibitor of angiogenesis. The possible role of apo(a) in microvascular proliferation was studied in an in vivo quantitative model, the disc angiogenesis system (DAS) and compared to angiostatin. Apo(a) and other test compounds were placed in the center of a polyvinyl alcohol foam disc that was implanted subcutaneously in mice. After 14 days, the disc was removed and vascular growth into the disc was measured. Apo(a) did not affect spontaneous vessel growth into the disc, while angiostatin suppressed this growth and basic fibroblast growth factor (bFGF) increased it. Additionally, apo(a) did not modify the vascular growth induced by bFGF. Transgenic mice expressing the human apo(a) gene were used to study the systemic effect of apo(a): neither an increase nor a decrease in vascular growth was detected. Our results suggest that apo(a) is unlikely to play a significant role in the control of angiogenesis. Furthermore, our experiments confirm the inhibitory effect of angiostatin not only on induced angiogenesis but also on baseline, spontaneous angiogenesis.

High dose rate intracoronary radiation for inhibition of neointimal formation in the stented and balloon-injured porcine models of restenosis: angiographic, morphometric, and histopathologic analyses.

PURPOSE: We examined the effects of intracoronary irradiation delivered at a high dose rate on neointimal hyperplasia after injury induced by two methods: balloon overstretch injury, and stent implantation in a porcine model of coronary restenosis. METHODS AND MATERIALS: In 34 Hanford miniature swine, a segment of each coronary artery was targeted for injury and treatment. The artery segments were treated with 192Ir at doses of 10 Gy over 4 min (eight animals), 15 Gy over 6 min (nine animals), 25 Gy over 10 min (nine animals) or control (simulation wire only; eight animals). The treated segments were subjected to stent implantation (left anterior descending and right coronary artery) or balloon overstretch (circumflex) injury. Twenty-eight days later, repeat coronary angiography and sacrifice were done. Quantitative coronary angiography, morphometry, and extensive histopathologic analyses were carried out in a blinded fashion. RESULTS: The change in minimal lumen diameter from postinjury to presacrifice in the stent-injured left anterior descending was -0.79 +/- 0.34 (mean: +/- SD) mm in the control group, compared to -0.43 +/- 0.35 mm in the 15 Gy (p = 0.04) and -0.21 +/- 0.50 mm in the 25 Gy (p = 0.01) groups; and in the balloon-injured circumflex was -0.31 +/- 0.22 mm in the control group compared to -0.03 +/- 0.18 mm in the 10 Gy (p = 0.05) and 0.00 +/- 0.33 in the 15 Gy (p = 0.01) groups. Percent area stenosis in the left anterior descending was 36 +/- 9% in the control group compared to 18 +/- 12% in the 15 Gy (p = 0.003) and 11 +/- 11% in the 25 Gy (p < 0.001) groups; and in the circumflex was 16 +/- 10% in the control groups, compared to 5 +/- 5% in the 15 Gy (p = 0.02) and 2 +/- 2% in the 25 Gy (p = 0.009) groups. Histopathology showed a striking reduction in the amount of neointima in the irradiated arteries compared with control vessels. Other radiation effects were stromal fibrin exudate, thinning of the media, and adventitial fibrosis and leukocyte infiltration in the radiated arterial segments. CONCLUSIONS: High dose rate intracoronary irradiation with 192Ir effectively inhibits intimal proliferation after stent-induced as well as balloon-overstretch injury. This shorter treatment time (4 to 10 min) may provide a clinically practical approach to the prevention of restenosis after angioplasty.

Lethal infection by a previously unrecognised metazoan parasite.

BACKGROUND: New microbial pathogens or variant clinical manifestations of known organisms may be first found in immunodeficient patients. An HIV-infected man developed a rapidly-enlarging abdominal mass, suggestive of a neoplasm, that subsequently invaded his liver and caused death. Initial studies showed unusual tissue morphology that could not be matched with any known disease process. METHODS: Tissues obtained from biopsy at laparotomy and necropsy were studied by light microscopy, immunohistochemistry, electron microscopy, and broad-range ribosomal DNA-amplification and sequence analysis. FINDINGS: Tissue lesions were characterised by peculiar cytoplasmic sacs containing minute cells with very prominent nucleoli. The pathological process was recognised as a parasitic infection, although its features were different from those of any known eukaryotic pathogen. Phylogenetic analysis of a 357 bp 18S rDNA sequence amplified directly from the involved tissue indicated that the causative agent was a previously-uncharacterised cestode. INTERPRETATION: Fatal disease produced by this newly recognised cestode may not be limited to immunodeficient hosts. Awareness of this metazoan infection may allow early diagnosis-by morphology and DNA sequence analysis--and perhaps successful treatment of subsequent cases.

Transforming growth factor beta1 induces angiogenesis in vivo with a threshold pattern.

SUMMARY: The true role of transforming growth factor beta1 (TGFbeta) on angiogenesis is in question. Several in vitro studies have shown inhibition of proliferation and migration of endothelial cells (EC). However, some investigators have observed that TGFbeta stimulates the formation of EC tubes in vitro. Fewer in vivo studies have been performed, but these also show discrepancies: some report angiogenic induction and at least one reports inhibition. We used the disc angiogenesis system (DAS) to measure the in vivo effect of TGFbeta. Discs containing 1 ng to 2000 ng of TGFbeta were placed subcutaneously in mice, removed after a growth period of 14 days, measured by three different techniques, and compared with spontaneous growth controls and with positive controls containing prostaglandin El. Tritiated thymidine was used to determine proliferation of EC. The discs were also examined morphologically for patterns of vessel and stromal proliferation. The contribution of native TGFbeta to the spontaneous angiogenesis of wound healing was tested using a monoclonal anti-TGFbeta antibody. The combined effect of TGFbeta and fibroblast growth factor (bFGF) was studied by using suboptimal doses of both (500 ng and 10 microg, respectively). Although TGFbeta doses of 1 ng to 500 ng failed to induce angiogenesis, 1000 ng induced a significant level of angiogenesis which was maintained at 2000 ng. This effect was the same regardless of the method of quantification: centripetal growth of the vessels, size of fibrovascular growth area, or amount of incorporation of tritiated thymidine. The anti-TGFbeta antibody decreased the spontaneous vascular growth below the level of controls containing irrelevant IgG. The combination of TGFbeta and bFGF at suboptimal doses did not increase or decrease the angiogenic response. Discs containing TGFbeta showed more collagen and greater accumulation of neutrophils than control discs or discs containing other cytokines. In conclusion, TGFbeta1 is angiogenic in vivo, when it reaches a threshold of 1 microg, but is not angiogenic at doses of 1 to 500 ng. Endogenous TGFbeta contributes to spontaneous (wound healing) angiogenesis. At the suboptimal doses of TGFbeta and bFGF used, there is no evidence of a combined angiogenic effect. The angiogenic effect of TGFbeta is probably indirect, requiring recruitment of leukocytes that secondarily release angiogenic substances. This secondary effect may explain some of the discrepancies between the in vitro and in vivo effects of TGFbeta.

Multifocal heterogeneity in villin and Ep-CAM expression in Barrett's esophagus.

Barrett's esophagus (BE) is a metaplastic change of the squamous esophageal epithelium to columnar gastric or intestinal-like epithelium. BE is associated with long-standing gastroesophageal reflux disease and carries an increased risk for dysplasia and adenocarcinoma. Little if any is known regarding the differentiation state of esophageal metaplasia and its relationship to carcinogenesis. In this study, we investigated the potential of villin, a cytoskeletal protein, and Ep-CAM, a glandular epithelial glycoprotein, to serve as markers for enterocytic differentiation in BE at the molecular level. Endoscopic mucosal biopsy samples of normal esophagus, BE, stomach and duodenum were collected from 23 patients with BE. Biopsies were analyzed for villin and Ep-CAM expression by immunoblotting, and in some cases for the presence of microvilli by electron microscopy. By mapping of BE segments in 6 patients, correlations were also made between the histologic evidence of metaplasia and villin expression. Villin was uniformly expressed in all duodenal samples but was not detected in normal esophagus and stomach. In BE biopsies, villin expression was limited to the subset of patients whose adjacent biopsies showed microvilli by electron microscopy. In several patients studied, however, the expression of villin and the epithelial glycoprotein Ep-CAM differed among various regions of esophageal metaplasia within the same patient. Mapping studies failed to reveal any correlation among histologic evidence of metaplasia, dysplasia and villin expression and confirmed the multifocal heterogeneity of villin expression in BE. Preliminary data of 4 adenocarcinoma patients studied showed that villin expression was absent in 3 and very low in 1 patient. Ep-CAM was highly expressed in all adenocarcinoma patients. Our results show that BE represents a complex epithelium with significant heterogeneity in antigen expression and ultrastructural morphologic features. This molecular heterogeneity supports the presence of different stages of differentiation within the same epithelium.

Dosimetry studies utilizing the urolase right-angle firing neodymium:YAG laser fiber in the human prostate.

BACKGROUND AND OBJECTIVE: Until recently, little or no objective data have been available to support either the choice of power setting or the timing of laser applications to achieve optimal tissue ablation in the human prostate. The objective of this study was to define quantitative dosimetry curves for the Urolase right angle laser fiber in human prostates. STUDY DESIGN MATERIALS AND METHODS: Transurethral Neodymium:YAG laser application was performed with the Urolase right-angle laser fiber in adult human prostates prior to planned radical surgery. Depth and volume of prostatic tissue coagulation for single, continuous laser applications were measured at variable power settings from 20 to 60 watts while holding total energy delivery constant. Then, holding the power setting constant at 40 watts, the extent of tissue coagulation was measured for variable treatment times from 60 to 120 seconds. RESULTS: Peak tissue coagulation was observed at 40 watts up to a maximum of 14 mm tissue penetration and 4.23 cc volume coagulated following a single spot laser application. The mean depth of tissue coagulation at 40 watts power setting was 13.5 mm, with a mean volume of tissue coagulation of 3.68 cc. The mean depth of tissue penetration at 40 watts was more than 25% greater than that observed at 60 watts, and the mean volume of tissue coagulation was 190% greater than that observed at 60 watts. As treatment time was increased from 60 to 90 seconds, extent of tissue coagulation increased significantly. However, beyond 90 seconds continuous laser application at 40 watts, a plateau in tissue effects was observed, with minimal increase in tissue coagulation between 90 and 120 seconds. Histologic examination of prostates removed acutely showed heat-induced damage to both stromal and glandular epithelial elements in laser-treated areas. At 1 year, the prostatic urethra was lined with a normal transitional epithelium, and mild periurethral fibrosis with focal squamous metaplasia was seen. CONCLUSION: Using the Urolase right-angle laser fiber, this study suggests that 40 watts power setting and 90 seconds continuous application time with a Neodymium:YAG laser source represent optimal treatment parameters to maximize prostatic tissue coagulation.

Radiation injury to the heart.

For the RTOG Consensus Conference on Late Effects of Cancer Treatment we summarize the clinical manifestations of cardiac complications appearing months to years following incidental irradiation of the heart during treatment of thoracic neoplasms. The most common effects present as pericardial disease, however, it is becoming more clear that precocious or accelerated coronary artery disease is an important late effect, especially in patients treated with radiation before the age of 21 years. To the extent it is known, the pathophysiology of the various syndromes is described and the extensive literature on dose, volume, and fractionation factors is reviewed. Based upon our current understanding of late cardiac effects, a clinical grading system has been developed and is published elsewhere in this issue.

Hepatic toxicity resulting from cancer treatment.

Radiation-induced liver disease (RILD), often called radiation hepatitis, is a syndrome characterized by the development of anicteric ascites approximately 2 weeks to 4 months after hepatic irradiation. There has been a renewed interest in hepatic irradiation because of two significant advances in cancer treatment: three dimensional radiation therapy treatment planning and bone marrow transplantation using total body irradiation. RILD resulting from liver radiation can usually be distinguished clinically from that resulting from the preparative regime associated with bone marrow transplantation. However, both syndromes demonstrate the same pathological lesion: veno-occlusive disease. Recent evidence suggests that elevated transforming growth factor beta levels may play a role in the development of veno-occlusive disease. Three dimensional treatment planning offers the potential to determine the radiation dose and volume dependence of RILD, permitting the safe delivery of high doses of radiation to parts of the liver. The chief therapy for RILD is diuretics, although some advocate steroids for severe cases. The characteristics of RILD permit the development of a grading system modeled after the NCI Acute Common Toxicity Criteria, which incorporates standard criteria of hepatic dysfunction.

Endothelial cells and hyperthermia.

Most radiation oncologists are aware of the effects of clinical hyperthermia on neoplastic cells. Its effects on blood vessels, however, are not as well recognized. Yet, since the 1960s a number of investigators have described and categorized the effects of hyperthermia on microvessels (in vivo), and on cultured endothelial cells (EC) (in vitro). Both EC and microvessels can be lethally damaged by the hyperthermia doses used as antineoplastic therapy. In vitro data indicate that capillary EC are moderately sensitive to hyperthermia. Proliferating EC are more thermosensitive suggesting that microvessels of malignant neoplasms (which contain many proliferating EC) are more affected than microvessels of normal tissues. This differential sensitivity of microvessels has also been observed in blood flow studies. Furthermore, hyperthermia inhibits angiogenesis. Thus, some of the antineoplastic effects of heat are caused by ischaemia due to obstruction or destruction of the tumour vessels or to inability to form new vessels. Sublethal EC damage can also be demonstrated, resulting in decreased synthesis of most proteins including adhesion molecules (as well as increased expression of a few such as heat shock proteins) and producing reversible loss of cytoskeletal elements. The therapeutic advantage provided by the higher thermal sensitivity of neoplastic vessels should be exploited further, perhaps by developing strategies specifically aimed to the tumour microvasculature.