Role of TGF Beta and PPAR Alpha Signaling Pathways in Radiation Response of Locally Exposed Heart: Integrated Global Transcriptomics and Proteomics Analysis.

Epidemiological data from patients undergoing radiotherapy for thoracic tumors clearly show the damaging effect of ionizing radiation on cardiovascular system. The long-term impairment of heart function and structure after local high-dose irradiation is associated with systemic inflammatory response, contraction impairment, microvascular damage, and cardiac fibrosis. The goal of the present study was to investigate molecular mechanisms involved in this process. C57BL/6J mice received a single X-ray dose of 16 Gy given locally to the heart at the age of 8 weeks. Radiation-induced changes in the heart transcriptome and proteome were investigated 40 weeks after the exposure. The omics data were analyzed by bioinformatics tools and validated by immunoblotting. Integrated network analysis of transcriptomics and proteomics data elucidated the signaling pathways that were similarly affected at gene and protein level. Analysis showed induction of transforming growth factor (TGF) beta signaling but inactivation of peroxisome proliferator-activated receptor (PPAR) alpha signaling in irradiated heart. The putative mediator role of mitogen-activated protein kinase cascade linking PPAR alpha and TGF beta signaling was supported by data from immunoblotting and ELISA. This study indicates that both signaling pathways are involved in radiation-induced heart fibrosis, metabolic disordering, and impaired contractility, a pathophysiological condition that is often observed in patients that received high radiation doses in thorax.

Mouse bone marrow-derived endothelial progenitor cells do not restore radiation-induced microvascular damage.

Background. Radiotherapy is commonly used to treat breast and thoracic cancers but it also causes delayed microvascular damage and increases the risk of cardiac mortality. Endothelial cell proliferation and revascularization are crucial to restore microvasculature damage and maintain function of the irradiated heart. We have therefore examined the potential of bone marrow-derived endothelial progenitor cells (BM-derived EPCs) for restoration of radiation-induced microvascular damage. Material & Methods. 16 Gy was delivered to the heart of adult C57BL/6 mice. Mice were injected with BM-derived EPCs, obtained from Eng(+/+) or Eng(+/-) mice, 16 weeks and 28 weeks after irradiation. Morphological damage was evaluated at 40 weeks in transplanted mice, relative to radiation only and age-matched controls. Results. Cardiac irradiation decreased microvascular density and increased endothelial damage in surviving capillaries (decrease alkaline phosphatase expression and increased von Willebrand factor). Microvascular damage was not diminished by treatment with BM-derived EPCs. However, BM-derived EPCs from both Eng(+/+) and Eng(+/-) mice diminished radiation-induced collagen deposition. Conclusion. Treatment with BM-derived EPCs did not restore radiation-induced microvascular damage but it did inhibit fibrosis. Endoglin deficiency did not impair this process.

ANTIDotE: anti-tick vaccines to prevent tick-borne diseases in Europe.

Ixodes ricinus transmits bacterial, protozoal and viral pathogens, causing disease and forming an increasing health concern in Europe. ANTIDotE is an European Commission funded consortium of seven institutes, which aims to identify and characterize tick proteins involved in feeding and pathogen transmission. The knowledge gained will be used to develop and evaluate anti-tick vaccines that may prevent multiple human tick-borne diseases. Strategies encompassing anti-tick vaccines to prevent transmission of pathogens to humans, animals or wildlife will be developed with relevant stakeholders with the ultimate aim of reducing the incidence of tick-borne diseases in humans.

Radiation- and anthracycline-induced cardiac toxicity and the influence of ErbB2 blocking agents.

In Her2-positive breast cancer patients, inhibition of epidermal growth factor receptor 2 (ErbB2)-signaling is often combined with chemotherapy and radiotherapy. The risk of cardiac toxicity after anthracyclines and radiotherapy is recognized, but little is known about increased risk when these treatments are combined with ErbB2 inhibition. This study investigated whether ErbB2 inhibition increased radiation or anthracycline-induced toxicity. In an in vitro study, human cardiomyocytes were treated with irradiation or doxorubicin, alone or in combination with trastuzumab, and evaluated for cell survival and growth. Groups of mice received 0 or 14 Gy to the heart, alone or in combination with lapatinib, or 3 × 4 mg/kg doxorubicin alone or in combination with lapatinib. Mice were evaluated 40 weeks after treatment for cardiac damage. Changes in cardiac function ((99m)Tc-Myoview gated SPECT) were related to histomorphology and microvascular damage. Radiation or doxorubicin-induced cardiomyocyte toxicity (in vitro) were not exacerbated by trastuzumab. Cardiac irradiation of mice decreased microvascular density (MVD) and increased endothelial damage in surviving capillaries (decrease alkaline phosphatase expression and increased von Willebrand factor), but these changes were not exacerbated by lapatinib. Inflammatory responses in the irradiated epicardium (CD45+ and F4/80+ cells) were significantly reduced in combination with lapatinib. Irradiation, doxorubicin, and lapatinib each induced cardiac fibrosis but this was not further enhanced when treatments were combined. At the ultra-structural level, both lapatinib and doxorubicin induced mitochondrial damage, which was enhanced in combined treatments. Lapatinib alone also induced mild changes in cardiac function but this was not enhanced in the combined treatments. Trastuzumab did not enhance direct radiation or anthracycline toxicity of cardiomyocytes in vitro. Lapatinib did not enhance the risk of radiation or anthracycline-induced cardiac toxicity in mice up to 40 weeks after treatment, but mitochondrial damage was more severe after doxorubicin combined with lapatinib.

Endoglin haplo-insufficiency modifies the inflammatory response in irradiated mouse hearts without affecting structural and mircovascular changes.

BACKGROUND: It is now widely recognized that radiotherapy of thoracic and chest wall tumors increases the long-term risk of cardiovascular damage although the underlying mechanisms are not fully elucidated. There is increasing evidence that microvascular damage is involved. Endoglin, an accessory receptor for TGF-ß1, is highly expressed in damaged endothelial cells and may play a crucial role in cell proliferation and revascularization of damaged heart tissue. We have therefore specifically examined the role of endoglin in microvascular damage and repair in the irradiated heart. MATERIALS & METHODS: A single dose of 16 Gy was delivered to the heart of adult Eng(+/+) or Eng(+/-) mice and damage was evaluated at 4, 20 and 40 weeks, relative to age-matched controls. Gated single photon emission computed tomography (gSPECT) was used to measure cardiac geometry and function, and related to histo-morphology, microvascular damage (detected using immuno- and enzyme-histochemistry) and gene expression (detected by microarray and real time PCR). RESULTS: Genes categorized according to known inflammatory and immunological related disease were less prominently regulated in irradiated Eng(+/-) mice compared to Eng(+/+) littermates. Fibrosis related genes, TGF-ß1, ALK 5 and PDGF, were only upregulated in Eng(+/+) mice during the early phase of radiation-induced cardiac damage (4 weeks). In addition, only the Eng(+/+) mice showed significant upregulation of collagen deposition in the early fibrotic phase (20 weeks) after irradiation. Despite these differences in gene expression, there was no reduction in inflammatory invasion (CD45+cells) of irradiated Eng(+/-) hearts. Microvascular damage (microvascular density, alkaline phosphatase and von-Willebrand-Factor expression) was also similar in both strains. CONCLUSION: Eng(+/-) mice displayed impaired early inflammatory and fibrotic responses to high dose irradiation compared to Eng(+/+) littermates. This did not result in significant differences in microvascular damage or cardiac function between the strains.

Thalidomide is not able to inhibit radiation-induced heart disease.

PURPOSE: Radiotherapy to the thorax increases the risk of radiation-induced heart disease. We and others have shown that local irradiation to the murine heart results in inflammatory and fibrotic responses and decreased microvascular density. In the present study we tested whether thalidomide is able to inhibit radiation-induced heart disease. METHODS: Single doses of 16 Gy or 0 Gy (sham treatment) were delivered to the hearts of mice. At 16 weeks after irradiation the mice were allocated to receive a thalidomide-containing chow (100 mg/kg body weight/day) or control chow until the end of the experiment. At 40 weeks after irradiation, functional imaging was performed and the hearts were examined for histological damage. RESULTS: Irradiation led to an increase in epicardial thickness and infiltrating inflammatory cells in the epicardium as well as an increase in interstitial collagen content. The microvasculature had a decreased alkaline phosphatase activity and reduced pericyte coverage. Thalidomide had no protective role in any of these processes. There were no differences in heart function measured between the treatment groups. CONCLUSIONS: Although others have shown protective effects of thalidomide in disease models involving inflammation, fibrosis and blood vessel maturation, thalidomide was not able to reduce radiation-induced heart damage.

Local heart irradiation of ApoE(-/-) mice induces microvascular and endocardial damage and accelerates coronary atherosclerosis.

BACKGROUND AND PURPOSE: Radiotherapy of thoracic and chest-wall tumors increases the long-term risk of radiation-induced heart disease, like a myocardial infarct. Cancer patients commonly have additional risk factors for cardiovascular disease, such as hypercholesterolemia. The goal of this study is to define the interaction of irradiation with such cardiovascular risk factors in radiation-induced damage to the heart and coronary arteries. MATERIAL AND METHODS: Hypercholesterolemic and atherosclerosis-prone ApoE(-/-) mice received local heart irradiation with a single dose of 0, 2, 8 or 16 Gy. Histopathological changes, microvascular damage and functional alterations were assessed after 20 and 40 weeks. RESULTS: Inflammatory cells were significantly increased in the left ventricular myocardium at 20 and 40 weeks after 8 and 16 Gy. Microvascular density decreased at both follow-up time-points after 8 and 16 Gy. Remaining vessels had decreased alkaline phosphatase activity (2-16 Gy) and increased von Willebrand Factor expression (16 Gy), indicative of endothelial cell damage. The endocardium was extensively damaged after 16 Gy, with foam cell accumulations at 20 weeks, and fibrosis and protein leakage at 40 weeks. Despite an accelerated coronary atherosclerotic lesion development at 20 weeks after 16 Gy, gated SPECT and ultrasound measurements showed only minor changes in functional cardiac parameters at 20 weeks. CONCLUSIONS: The combination of hypercholesterolemia and local cardiac irradiation induced an inflammatory response, microvascular and endocardial damage, and accelerated the development of coronary atherosclerosis. Despite these pronounced effects, cardiac function of ApoE(-/-) mice was maintained.

Irradiation induced modest changes in murine cardiac function despite progressive structural damage to the myocardium and microvasculature.

BACKGROUND: Radiotherapy of thoracic and chest wall tumors increases the long-term risk of cardiotoxicity, but the underlying mechanisms are unclear. METHODS: Single doses of 2, 8, or 16 Gy were delivered to the hearts of mice and damage was evaluated at 20, 40, and 60 weeks, relative to age matched controls. Single photon emission computed tomography (SPECT/CT) and ultrasound were used to measure cardiac geometry and function, which was related to histo-morphology and microvascular damage. RESULTS: Gated SPECT/CT and ultrasound demonstrated decreases in end diastolic and systolic volumes, while the ejection fraction was increased at 20 and 40 weeks after 2, 8, and 16 Gy. Cardiac blood volume was decreased at 20 and 60 weeks after irradiation. Histological examination revealed inflammatory changes at 20 and 40 weeks after 8 and 16 Gy. Microvascular density in the left ventricle was decreased at 40 and 60 weeks after 8 and 16 Gy, with functional damage to remaining microvasculature manifest as decreased alkaline phosphatase (2, 8, and 16 Gy), increased von Willebrand Factor and albumin leakage from vessels (8 and 16 Gy), and amyloidosis (16 Gy). 16 Gy lead to sudden death between 30 and 40 weeks in 38% of mice. CONCLUSIONS: Irradiation with 2 and 8 Gy induced modest changes in murine cardiac function within 20 weeks but this did not deteriorate further, despite progressive structural and microvascular damage. This indicates that heart function can compensate for significant structural damage, although higher doses, eventually lead to sudden death.

Regulation of direct transintestinal cholesterol excretion in mice.

Biliary secretion is generally considered to be an obligate step in the pathway of excess cholesterol excretion from the body. We have recently shown that an alternative route exists. Direct transintestinal cholesterol efflux (TICE) contributes significantly to cholesterol removal in mice. Our aim was to investigate whether the activity of this novel pathway can be influenced by dietary factors. In addition, we studied the role of cholesterol acceptors at the luminal side of the enterocyte. Mice were fed a Western-type diet (0.25% wt/wt cholesterol; 16% wt/wt fat), a high-fat diet (no cholesterol; 24% wt/wt fat), or high-cholesterol diet (2% wt/wt), and TICE was measured by isolated intestinal perfusion. Bile salt-phospholipid mixtures served as cholesterol acceptor. Western-type and high-fat diet increased TICE by 50 and 100%, respectively. In contrast, the high-cholesterol diet did not influence TICE. Intestinal scavenger receptor class B type 1 (Sr-B1) mRNA and protein levels correlated with the rate of TICE. Unexpectedly, although confirming a role for Sr-B1, TICE was significantly increased in Sr-B1-deficient mice. Apart from the long-term effect of diets on TICE, acute effects by luminal cholesterol acceptors were also investigated. The phospholipid content of perfusate was the most important regulator of TICE; bile salt concentration or hydrophobicity of bile salts had little effect. In conclusion, TICE can be manipulated by dietary intervention. Specific dietary modifications might provide means to stimulate TICE and, thereby, to enhance total cholesterol turnover.