Effect of N-acetyl cysteine and alpha-linolenic acid on sulfur mustard caused impairment of in vitro endothelial tube formation.

Sulfur mustard (SM), an alkylating chemical warfare agent, leads to tissue damage, including inflammation, blister formation, and impaired wound healing. Especially wound healing is of concern because after SM exposure, wound healing is prolonged. In this study, we focused on the effect of SM (30 and 100μM) on endothelial tube formation, apoptosis, and proliferation in mouse embryoid bodies (EBs), which provide an appropriate model for investigating vasculogenesis and angiogenesis. EBs were exposed to SM for 30 min on day 0, 3, or 6 of EBs' growth, were allowed to grow until day 7, then fixed, and immunostained (PECAM-1, Ki67, and activated caspase-3). SM significantly decreased endothelial tube formation compared with unexposed EBs. Additionally, we observed a significant increase of apoptosis. As the formation of reactive oxygen species (ROS) is discussed to be involved in the pathophysiology of SM toxicity, we evaluated the effect of ROS scavengers (α-linolenic acid [ALA] and N-acetyl cysteine [NAC]) in the same experimental setup. Temporary effects of both scavengers could be detected, in particular NAC seemed to have temporary significant positive effects on endothelial tube formation in 100μM SM-exposed EBs. ALA augmented proliferation when administered after 30μM SM exposure on day 3, whereas NAC treatment on day 0 decreased apoptosis induced by 100μM SM. Taken together, our findings pointed to a negative effect of SM on vascularization and endothelial tube formation. ROS scavengers NAC and ALA showed temporary, but not long-lasting, rescuing effects regarding endothelial tube formation after SM exposure.

[1]  R Kemler,et al.  The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. , 1985, Journal of embryology and experimental morphology.

[2]  C. Kunsch,et al.  Oxidative stress as a regulator of gene expression in the vasculature. , 1999, Circulation research.

[3]  R. Timpl,et al.  Endostatin influences endothelial morphology via the activated ERK1/2-kinase endothelial morphology and signal transduction. , 2006, Microvascular research.

[4]  W. Bloch,et al.  Sulphur mustard induces time- and concentration-dependent regulation of NO-synthesizing enzymes. , 2009, Toxicology letters.

[5]  G. Kojda,et al.  Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice. , 2005, American journal of physiology. Heart and circulatory physiology.

[6]  R. Schwinger,et al.  Role of erythropoietin for angiogenesis and vasculogenesis: from embryonic development through adulthood. , 2006, American journal of physiology. Heart and circulatory physiology.

[7]  Bernadette Ateghang,et al.  Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain‐induced cardiovascular differentiation , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[8]  R. Jain,et al.  Role of eNOS in neovascularization: NO for endothelial progenitor cells. , 2004, Trends in molecular medicine.

[9]  I. Thorey,et al.  Endostatin down-regulates soluble guanylate cyclase (sGC) in endothelial cells in vivo: influence of endostatin on vascular endothelial growth factor (VEGF) signaling. , 2005, Endothelium : journal of endothelial cell research.

[10]  M. Naghii.  Sulfur mustard intoxication, oxidative stress, and antioxidants. , 2002, Military medicine.

[11]  W. Bloch,et al.  Nitrogen mustard (Chlorambucil) has a negative influence on early vascular development. , 2009, Toxicology.

[12]  W. Risau,et al.  Molecular mechanisms of vasculogenesis and embryonic angiogenesis , 1997, Journal of cellular physiology.

[13]  H. Hassankhani,et al.  The challenges experienced by Iranian war veterans living with chemical warfare poisoning: a descriptive, exploratory study. , 2010, Scandinavian journal of caring sciences.

[14]  J. Isner,et al.  Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. , 1999, Circulation research.

[15]  Jie Li,et al.  Angiogenesis in wound repair: Angiogenic growth factors and the extracellular matrix , 2003, Microscopy research and technique.

[16]  K. Kehe,et al.  Molecular toxicology of sulfur mustard-induced cutaneous inflammation and blistering. , 2009, Toxicology.

[17]  Z. Suntres,et al.  Sulfur Mustard Toxicity Following Dermal Exposure , 2007, Journal of burns and wounds.

[18]  H. DeLisser,et al.  Molecular and functional aspects of PECAM-1/CD31. , 1994, Immunology today.

[19]  H. Mortazavi,et al.  Late cutaneous manifestations 14 to 20 years after wartime exposure to sulfur mustard gas: a long-term investigation. , 2008, Archives of dermatology.

[20]  Stephen M Bauer,et al.  Angiogenesis, Vasculogenesis, and Induction of Healing in Chronic Wounds , 2005, Vascular and endovascular surgery.

[21]  M. Wolin.  Reactive oxygen species and the control of vascular function. , 2009, American journal of physiology. Heart and circulatory physiology.

[22]  T. Doetschman,et al.  Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. , 1988, Development.

[23]  C. Garlanda,et al.  Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic endothelium. , 1994, European journal of cell biology.

[24]  P. Albertsson,et al.  Chemotherapy and antiangiogenesis: drug-specific effects on microvessel sprouting. , 2003, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[25]  Mark Denham,et al.  Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture and Differentiation as Embryoid Bodies , 2005, Current protocols in cell biology.

[26]  D. T. Robles,et al.  Keloids: pathophysiology and management. , 2007, Dermatology online journal.

[27]  M. Mahmoudi,et al.  Delayed complications of sulfur mustard poisoning in the skin and the immune system of Iranian veterans 16–20 years after exposure , 2006, International journal of dermatology.

[28]  J. Kreuger,et al.  Building blood vessels—stem cell models in vascular biology , 2007, The Journal of cell biology.

[29]  William J Smith,et al.  Cytometric analysis of DNA changes induced by sulfur mustard , 1993 .

[30]  P. Schlag,et al.  [The surgical treatment of patients treated with cytostatic chemotherapy]. , 1989, Der Chirurg; Zeitschrift fur alle Gebiete der operativen Medizen.

[31]  D. Palanker,et al.  Early nonsurgical removal of chemically injured tissue enhances wound healing in partial thickness burns. , 1998, Burns : journal of the International Society for Burn Injuries.

[32]  R. Jain,et al.  Vascular Morphogenesis and Remodeling in a Model of Tissue Repair: Blood Vessel Formation and Growth in the Ovarian Pedicle After Ovariectomy , 2001, Circulation research.

[33]  D. Hinshaw,et al.  Sulfur mustard induces apoptosis and necrosis in endothelial cells. , 1996, Toxicology and applied pharmacology.

[34]  O. Féraud,et al.  Vasculogenesis and angiogenesis from in vitro differentiation of mouse embryonic stem cells. , 2003, Methods in enzymology.

[35]  D. Ribatti,et al.  Postnatal vasculogenesis , 2001, Mechanisms of Development.

[36]  P. Rice.  Sulphur Mustard Injuries of the Skin , 2003, Toxicological reviews.

[37]  M. Putt,et al.  The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia. , 2006, Journal of vascular surgery.

[38]  D. Harrison.  Oxidant Signaling in Vascular Cell Growth , Death , and Survival A Review of the Roles of Reactive Oxygen Species in Smooth Muscle and Endothelial Cell Mitogenic and Apoptotic Signaling , 2000 .

[39]  W. Risau,et al.  Mechanisms of angiogenesis , 1997, Nature.

[40]  AnnetteSchmidt,et al.  Endothelial Precursor Cell Migration During Vasculogenesis , 2007 .

[41]  C. White,et al.  A Role for Mitochondrial Oxidative Stress in Sulfur Mustard Analog 2-Chloroethyl Ethyl Sulfide-Induced Lung Cell Injury and Antioxidant Protection , 2009, Journal of Pharmacology and Experimental Therapeutics.