Death of human tumor endothelial cells in vitro through a probable calcium-associated mechanism induced by bevacizumab and detected via a novel method

We isolated three dimensional cell clusters from fresh human solid tumors and also isolated human neoplastic and normal lymphatic cells. Cells were cultured for 96 hours with and without bevacizumab and other agents. At concentrations of bevacizumab which completely removed VEGF from the culture medium, dead microvascular cells were detected through Fast Green/H&E staining as previously described. These peculiar staining characteristics suggested the involvement of calcium, and this was confirmed through staining with Alizarin red S. Using Alizarin staining as a marker for endothelial cell death permitted the use of public domain image analysis software which resulted in a sensitive and specific system for identifying active pharmaceuticals which target the tumor microvasculature at the same time direct antitumor cell effects are determined. Our results suggest an important role for calcium in endothelial cell death mediated by bevacizumab and other agents and further suggest that agents promoting calcium influx may potentiate the activity of antiangiogenic agents

Endothelial Massive Calcium Accumulation Death (MCAD): Mechanism, Target, and Predictive Biomarker for Anti-Angiogenic Therapy

We cultured human umbilical vein endothelial cells with bevacizumab, with tyrosine kinase inhibitors known to be AA, and with traditional cytotoxic drugs. The images below show that, in the presence of physiological saline and non-favorable culture conditions, the vast majority of the endothelial cells undergo a "non-specific" type of cell death (NSCD), not associated with calcium accumulation, but with loss of cell membrane integrity, allowing uptake of the Fast Green dye, staining these dead dells a pale blue green. In the presence of known AA agents (e.g. bevacizumab, some TK inhibitors) a large percentage of the endothelial cells undergo death associated with massive calcium accumulation (MCAD), with these cells staining hyperchromatic, refractile, blue-black, precisely as reported in http://www.ncbi.nlm.nih.gov/pubmed/18793333 and http://meeting.ascopubs.org/cgi/content/abstract/
28/15_suppl/e13617 and http://tinyurl.com/weisenthal-breast-lapatinib. MCAD is strikingly demonstrated by Fast Green/Alizarin staining as reported in http://precedings.nature.com/documents/4499/version/1. Traditional cytotoxic drugs (e.g. cisplatin) produce only NSCD and inhibit MCAD. We propose that MCAD is a cell death mechanism unique to endothelial cells and provides a practical biomarker to predict for AA activity in clinical oncology and drug development, as well as a potential drug target

Bevacizumab-induced tumor calcifications can be elicited in glioblastoma microspheroid culture and represent massive calcium accumulation death (MCAD) of tumor endothelial cells

Bähr and colleagues reported that 22 of 36 glioblastoma patients treated with bevacizumab showed tumor calcifications on 8 week post therapy follow up with MRI. Early tumor calcification strongly predicted for response, time to progression, and overall survival. The authors didn’t understand the mechanism, but speculated that it was vascular in nature. At the 13th International Anti-Angiogenic Symposium (2011), we presented our discovery of the phenomenon of massive calcium accumulation death, wherein MCAD occurred in endothelial cells (tumor, circulating, and HUVEC), in response to VEGF depletion by bevacizumab and other putative anti-angiogenic agents, but not in response to non-specific cytotoxins. In subsequent work, we have documented marked MCAD to occur in primary microcluster cultures from 6 fresh human glioblastoma biopsies, following 96 hours of VEGF depletion in vitro by bevacizumab. The presence and degree of MCAD is strikingly dependent on the type of serum in the culture medium (RPMI-1640 + 25% serum) -- typically most striking in (very low VEGF) fetal calf serum, but inhibited (often) or enhanced (rarely) by 25% human serum from different patients or normal donors containing variable quantities of VEGF. There was not a linear relationship between VEGF concentration and MCAD inhibition (or enhancement), suggesting that other pro-angiogenic (or anti-angiogenic) serum factors may play a role. In epithelial metastatic tumors, circulating peripheral blood endothelial cells may be easily tested, using our methods, and the serum inhibition (or, rarely, enhancement) is faithfully reproduced on circulating endothelial cells, in comparison with the tumor cluster-associated endothelial cells. We propose MCAD as the mechanism of glioblastoma calcification following bevacizumab and further propose that testing tumor microclusters and/or circulating endothelial cells, in the presence of autologous serum, could be a useful predictive biomarker and research tool

Massively calcified endosomal death (MCED) of endothelial cells

We have discovered a novel and specific mechanism of endothelial cell death.We refer to this novel death mechanism as massively calcified endosomal death, or MCED. Exposure of endothelial cells to non-specific toxins or other physical stresses induces death by traditional apoptotic and non-apoptotic mechanisms, common to most different types of cells. In contrast, exposure of endothelial cells (but not other types of nucleated cells) to specific insults, such as oxidized pathogenic lipids (e.g. 7-ketocholesterol) or agents with known anti-angiogenic activity (e.g. bevacizumab, certain tyrosine kinase inhibitors, etc.) triggers cell death via a novel pathway, which involves the formation of massively calcified endosomes, which, in turn, escape from the dying endothelial cells as massively calcified exosomes. These endosomes/exosomes appear capable of provoking an inflammatory response, characterized by physical association of calcified microparticles with inflammatory cells (monocytes, lymphocytes, neutrophils) with resulting increased release of an inflammatory mediator (TNF) into the culture medium. Traditional media for the culture of endothelial cells are profoundly inhibitory to MCED, as are some mammalian sera and many human sera, explaining why MCED had not been previously discovered and reported. The present discovery of MCED was accidental, resulting from work with primary cultures of fresh human tumor cell clusters, which invariably contain microcapillary cells Our culture media are optimized for the tumor cells and not for the endothelial cells and, thus, are permissive of MCED. I propose MCED as the central mechanism underlying both intimal calcification and vascular inflammation in atherosclerosis.</jats:p

Content And Catabolism Of Chromatin Proteins In Control And Phytohemagglutinin Stimulated Human Lymphocytes, Leukemic Leukocytes And Burkitt Lymphoma Cells.

PhDBiochemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/190831/2/7425357.pd

Death of human tumor endothelial cells in vitro through a probable calcium-associated mechanism induced by bevacizumab and detected via a novel method

AbstractWe isolated three dimensional cell clusters from fresh human solid tumors and also isolated human neoplastic and normal lymphatic cells. Cells were cultured for 96 hours with and without bevacizumab and other agents. At concentrations of bevacizumab which completely removed VEGF from the culture medium, dead microvascular cells were detected through Fast Green/H&amp;E staining as previously described. These peculiar staining characteristics suggested the involvement of calcium, and this was confirmed through staining with Alizarin red S. Using Alizarin staining as a marker for endothelial cell death permitted the use of public domain image analysis software which resulted in a sensitive and specific system for identifying active pharmaceuticals which target the tumor microvasculature at the same time direct antitumor cell effects are determined. Our results suggest an important role for calcium in endothelial cell death mediated by bevacizumab and other agents and further suggest that agents promoting calcium influx may potentiate the activity of antiangiogenic agents.</jats:p

Swimming's shoulder

Na década de 80 descobriu-se que a exposição crónica ao ruído de baixa frequência pode causar a Doença Vibroacústica (DVA), uma patologia sistémica causada pela exposição prolongada (>10 anos) a ruído de grandes amplitudes de pressão (≥90 dB Sound Pressure Level) e baixa frequência (≤500 Hertz, incluindo os infrasons). Caracteriza-se pela proliferação anormal das matrizes extra-celulares (colagénio e elastina) em resposta à ‘agressão’ causada pelo impacto e propagação das vibrações acústicas sobre as células (mecanotransdução). Afecta preferencialmente as estruturas cardiovasculares (espessamento do pericárdio e válvulas cardíacas), o aparelho respiratório e o SNC. O RBF foi inicialmente identificado em ambientes de indústria pesada, mas com os avanços da investigação, demonstrou-se que está presente em áreas urbanas, algumas áreas rurais, numerosos locais de trabalho e numa grande variedade de outros locais e actividades de lazer, representando um problema de Saúde Pública. Pretende-se sistematizar a informação existente sobre a Doença Vibroacústica e estudos desenvolvidos em modelos animais e seres humanos, no sentido de explicar e demonstrar os efeitos deletérios/nocivos do RBF sobre os tecidos biológicos. Como o RBF não é ‘audível’, ainda não é oficialmente considerado um agente patológico e, como tal, as medições acústicas previstas pela lei raramente têm em conta as frequências abaixo de 500Hz. Desta forma/Nesta sequência, pretende-se também analisar a evolução da legislação nesta área de Saúde Pública, tendo como referência DVA, enquanto doença profissional e o RBF, como agente causador. A investigação detalhada da Doença Vibroacústica está dependente do reconhecimento do RBF como agente patológico. Quando forem conhecidos os efeitos específicos dose-frequência, poderão desenvolver-se estudos epidemiológicas em larga escala, que permitam conhecer o verdadeiro impacto do RBF sobre a humanidade. Só então poderá ser reavaliado o papel da DVA como doença profissional, equacionando novas estratégias de prevenção e tratamento.In the late 80’s it was found that chronic exposure to low frequency noise can cause Vibroacoustic disease, a systemic disease caused by long-term exposure (> 10 years) to large amplitude (≥ 90 dB) and low frequency noise (≤ 500 Hertz, including infrasound). It is characterized by abnormal proliferation of extra-cellular matrices (collagen and elastin) in response to the aggression caused by the impact and propagation of acoustic vibrations throughout the cells (mechanotransduction). It preferentially affects the cardiovascular structures (thickening of the pericardium and heart valves), respiratory tract and CNS. Low frequency noise was first identified in heavy industry environments, but with the advances in research, it was also demonstrated in other workplaces, urban areas, some rural areas and many leisure activities, representing a public health problem. The aim of this paper is to systematize the data on Vibroacoustic Disease, experiments in animal models and humans, in order to explain and demonstrate the effects of RBF on biological tissues. Since low frequency noise is not audible, it still has not been officially considered as a pathological agent. As such, noise measurements provided by Portuguese law rarely take into account the frequencies below 500Hz. Subsequently, this paper also intends to analyze the evolution of legislation in this area of Public Health, with reference to Vibroacoustic Disease as an occupational disease and low frequency noise as the causative agent. Further investigation upon Vibroacoustic Disease depends on the recognition of low frequency noise as a harmful agent. Further knowledge about the acoustical spectrum and specific dose-effects may provide the scientific basis to the development of wide-range epidemiological studies and better understanding of the true impact of low frequency noise on humanity. Only then the role of Vibroacoustic Disease as an occupational disease can be re-evaluated, new strategies for prevention and treatment can be re-equated