Manuela Martins-Green

1) Normal healing processes. In the early stages of my career, I pioneered investigation of the role of chemokines in wound healing. Chemokines are small, secreted, stress-response cytokines that are highly conserved among higher vertebrates and are now known to be important in inflammatory diseases, viral infections (e.g. HIV), fibrosis and tumorigenesis. As my program has developed, I have concentrated on activation, expression, function and mode of action of chemokines in healing, using animal models and complex human cultures (see #3 below). Our studies have focused on the chemokine, Interleukine-8 (IL-8), what agents at the wound site stimulate its expression, the signal-transduction mechanisms by which this expression occurs, and what functions IL-8 performs during the healing process. Our aim is to: (a) Identify commonalities in the signal transduction and transcription activation mechanisms that may lead the way to regulating the expression of chemokines for potential medical applications; (b) test the effects of IL-8 on the principal cellular components of the granulation tissue of wounds -- fibroblasts, myofibroblasts, endothelial cells, keratinocytes and immune cells - accompanied by tests directly to wounds; (c) characterize the function of IL-8 receptor(s) during wound healing.

2) Impaired healing. I have focused on the effects of environmental toxicants, especially those present in cigarette smoke. We investigate the effects of (primarily) second-hand smoke on: (a) the development of atherosclerotic plaques (a form of impaired healing of blood vessels); (b) skin wound healing; (c) on cornea epithelial wound healing. I am particularly interested in how chemokines are involved in these processes. Our aim here is to identify key processes affected by cigarette smoke and which chemicals in the smoke cause those effects. The extreme complexity of cigarette smoke has frustrated efforts to identify critical harmful components. We have now established a mouse model that mimics the common human conditions that lead atherogenesis. We are using this model and are developing a human assay system (see #4) to identify the smoke components (or groups of components) responsible for initiation of atherosclerotic plaque formation. More recently we have developed a chronic wound models using genetic manipulations which lead to delayed wound healing and when infection ensues leads to a chronic wounds.

3) Bench to bedside. We are using muliple factors to treat wounds in a timely manner to discover combinations that not only improve healing but also the regenerative capacity of the tissue. We are particularly interested in developing treatments for large burns. Through collaborations, we are developing matrices that are biocompatible, can be applied in large areas, and are biodegradable -- either naturally or by applying an enzyme.

4) Engineering of human tissues. In order to test the relevance for human biology of some of the hypotheses we have developed in animal systems, we are developing complex tissue cultures using primary human cells. We have already developed a novel human "skin" organ culture that we are now using to study some of the fundamental cell and molecular processes in normal and abnormal healing. We have also developed a system that allows us to test the effects of inflammatory agents on endothelial/epithelial permeability and we are currently developing an arterial wall model system for studies of atherogenesis induced by first- and second-hand cigarette smoke. We have also engineered a mouse to express CXCR1 the specific receptor for IL-8 that is not functional in mice to be able to address the biology of IL-8 in this species. This transgene is conditionally expressed when the mice are crossed with the appropriate Cre mice and the construct contain many advantages that allow detection of cells in vivo and isolation of these cells for tissue to perform studies in culture.

• Martins-Green, M. (1988). Origin of the dorsal surface of the neural tube by progressive delamination of epidermal ectoderm and neuroepithelium: Implications for neurulation and neural tube defects. Development 103: 687-706. In this seminal paper as a graduate student, I proposed a novel way by which the neural tube develops and closes and the potential consequences for Spina bifida. My model was highlighted in a 1990 comprehensive review of mechanisms of neurulation as one of two important new ideas breaking ground on this subject and has been included in Developmental Biology textbooks.



• Martins-Green, M., M.J. Bissell (1990). Localization of 9E3/CEF4 in avian tissues: Expression is absent in RSV-induced tumors but is stimulated by injury. J. Cell Biol. 110: 581-595. (photo on journal cover). In this paper we discovered that tumors that develop due to viral infection do not necessarily retain components of the virus that can be detected in diagnostic assays. This was at the time a very unconventional proposition. Furthermore, we showed for the first time that chemokines are involved in response to injury caused by the tumor and in normal response to injury, in particular in the development of microvessels.



• Martins-Green, M., N. Boudreau, M.J. Bissell (1994). Inflammation is responsible for the development of wound tumors in RSV-infected newly-hatched chicks. Cancer Res. 54:4334-4341. In this publication we showed that Rous-Sarcoma-Virus (RSV) -induced tumors were dependent on inflammation and proposed that during inflammation macrophages play a key role for viral-induced tumor development and that this type of inflammation is also instrumental in HIV setting up of Aids because both injury and macrophages are critical for development of this disease.



• Feugate, J.E., QJ Li, S. Lu, M. Martins-Green (2001). The CXC chemokine cCAF stimulates differentiation of fibroblasts into myofibroblasts and accelerates wound closure in vivo. Journal of Cell Biology 156:161-172. This study showed for the first time that chemokines stimulate another process of wound healing (contraction of the wound) in addition to angiogenesis, in this manner making solid the contributions of these small cytokines to wound healing, something that was not known previously. We obtained a patent for these findings.



• Li, Q-J., SH Yang, Y. Maeda, FM Sladek, AD Sharrocks, M. Martins-Green (2003). Map kinase phosphorylation-dependent activation of Elk-1 leads to activation of the coactivator p300. EMBO Journal 22(2): 1-11. The work presented here made new and significant experimental and conceptual contributions to the field of transcription. We identified novel interactions between transcription factor and co-activator that could play a critical role in chromatin remodeling and gene activation. This mechanism may be important in regulation of expression of immediate early response genes, in particular those involved in stress responses.



• Li, QJ, M. Yao, W. Wong, V. Parpura, M. Martins-Green. (2004). The N- and C-terminal peptides of hIL8/CSCL8 are ligands for hCXCR1 and hCXCR2. FASEB, 10:10961/fj.02-1175fje. Three page summary published in. FASEBJ 18:776-778, 2004. In this paper we used the chemokine IL-8/CXCL8 and discovered that multifunctionality of chemokines is related to the function of peptides that represent not only the N-terminus but also the C-terminus of these highly conserved molecules and that their functions lead to different outcomes. Up to this time only the N-terminus of chemokines was thought to have functionality.



• Martins-Green, M., Q-J Li and Min Yao (2004). Engineering human skin in culture using primary adult cells. FASEB Dec 9, [Epub]. Here we show a new generation of organ cultures that mimics human skin. This system can help answer fundamental biological and medical questions and can potentially be developed to help with impaired healing. These procedures will help with development of more complex tissue and organ cultures prepared with adult human primary cells for studies of disease, testing of drugs, and potential application as replacement organs. This type of construction is now one of the topics for the TR01 RFAs at NIH.



• Petreaca, M., M. Yao, C. Ware, and M. Martins-Green (2008). VEGF promotes macrophage apoptosis through stimulation of tumor necrosis factor superfamily member 14 (Tnfsf14/Light). [Winner of a Young Investigator Award in 2006]. Wound Repair and Regeneration, 16:602-614. In this work we challenged the established paradigm that VEGF is a survival factor by demonstrating that it stimulates macrophage death during inflammation in vivo adn that this process of resolution of inflammation occurs through LIGHT.



• Yuan, H., J. Y.-J Shyy, and M. Martins-Green (2009). Second-Hand Smoke Stimulates Lipid Accumulation in the Liver by Modulating AMPK and SREBP-1. Journal of Hepatology 51:535-547. The journal solicited an extensive editorial (reference follows this paragraph) that emphasized the fundamental importance of our work. More than 40 news articles were published about this work worldwide. We showed that cigarette smoke inhibits AMPK function which, in turn, releases the brakes on the transcription factor SREBP which then turns on lipid synthesis. The lipid accumulates in the liver causing non-alcoholic fatty liver disease or NAFLD. This disease leads to non-alcoholic steatohepatitis (inflammation of the liver), followed by cirrhosis and eventually liver failure.



• Dai, Q., H. Yuan, M. Yao, N. Zou, and M. Martins-Green (2009). A 3-D Multi-Cellular Human Arterial Wall Culture System for the Study of Blood Vessel Biology and Disease. Submitted. The work in both 9 and 10 describes the development of an arterial wall for studies on the effects of second hand cigarette smoke (CS)toxins on initiation of atherosclerotic plaque development. This system will be useful to study initiation of plaque formation. Monocytes that are put in contact with the culture penetrate the wall culture much more efficiently, and become macrophages, when in the presence of CS. When exposed to CS and Oxi LDL a "plaque" forms with macrophages that are full of lipid. A poster presented previous to this submission won an award for innovation.