Research Interests

Klaus Ley, M.D., Professor of Biomedical Engineering, Molecular Physiology and Biological Physics

Research Interests:
1. Molecular mechanisms of atherosclerosis
2. Biomechanics of leukocyte adhesion and targeted ultrasound contrast agents
3. Molecular mechanisms of inflammation in Crohn's disease
4. Molecular mechanisms of neutrophil recruitment to the lung
5. Neutrophil homeostasis and proliferation in inflammation
 

Techniques in Use:
1. Intravital microscopy of transgenic and knockout mice
2. Cell culture of primary blood and endothelial cells (mouse and human)
3. Flow cytometry, including 8-color and Amnis
4. Mouse aortic isolation, pinning, histology, immunostaining
5. Immunostaining for adhesion molecules, chemokines, cytokines
6. Isolated-perfused mouse carotid artery
7. Bone marrow transplantation
8. Knockout and transgenic mice
9. Various biochemistry methods


1. Molecular mechanisms of atherosclerosis

One area of research is atherosclerosis, which is supported by NIH R01 HL 58108 (funded until 2010) and NIH P01 HL 55798 (funded until 2011). We have developed a model in which monocyte adhesion can be directly observed in carotid or femoral arteries isolated from apolipoprotein E knockout mice to identify the molecular mechanisms of monocyte recruitment. This model has been instrumental at identifying adhesion molecules and, more recently, chemokines that promote monocytes arrest. If a particular molecule appears to be important in this mechanistic model, we test its impact in a model using wire-induced injury to carotid arteries of apoE-/- mice and/or generate mice with multiple gene defects (for example, apoE-/- P-selectin-/-). Recently, we have developed a flow-cytometry based technique to more fully understand the immune cell composition of the normal atherosclerotic artery wall. Other recent discoveries include the causative role of P-selectin-dependent platelet-endothelial interactions for the development and progression of atherosclerotic lesions and work into the role of 12/15-lipoxygenase in lesion development.

Huo, Y., Schober, A., Forlow, S.B., Smith, D.F., Hyman, M.C., Jung, S., Littman, D.R., Weber, C., Ley, K. (2003). Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nature Medicine 9: 61-67.

Huo, Y., Zhao, L., Hyman, M.C., Shashkin, P., Harry, B.L., Clarke, S., Burcin, T., Smith, D.F., Forlow, B.F., Srinivasan, S., Stark, M., Hedrick, C.C., Pratico, D., Witztum, J.L., Nadler, J.L., Funk, C., Ley, K.  (2004). Critical role of macrophage 12/15-lipoxygenase for atherosclerosis in apolipoprotein E deficient mice. Circulation, 110:  2024-2031.

Galkina, E., Kadl, A., Sanders, J., Varughese, D., Sarembock, I.J., Ley, K. (2006). Constitutive lymphocyte recruitment into the aortic wall precedes development of atherosclerosis. J. Exp. Med. 203: 1273-1282

 

2. Biomechanics of leukocyte adhesion and application to targeted ultrasound contrast agents

This area of research is funded by NIH R01 EB002185 (funded until 2011). The focus of this research is on understanding how leukocytes roll on and adhere to vascular endothelium. Recent discoveries in this area include the hemodynamic impact of the endothelial surface layer (ESL), which essentially causes wall shear rate and wall shear stress on the endothelial surface to be equal to zero. Instead, the highest wall shear stress is experienced at the interface between the free lumen and the ESL, which extends about 0.5 mm into the lumen from the endothelial plasma membrane. This discovery fundamentally alters the understanding of leukocyte adhesion under flow. Recent work also focuses on the importance of leukocyte deformation for sustained, stable rolling. Applications of the insights obtained are used to construct better ultrasound contrast agents for molecular imaging.

Smith, M.L., Long, D.S. Damiano, E.R., Ley, K. (2003). Near-wall micro-PIV reveals a hydrodynamically relevant endothelial surface layer in venules in vivo. Biophys. J. 85: 637-645.

Takalkar, A.M., Klibanov, A.L., Rychak, J.J., Lindner, J.R., Ley, K. (2004). Binding and detachment dynamics of microbubbles targeted to P-selectin under controlled shear flow. J Control Release 96: 473-482.

 

3. Molecular mechanisms of inflammation in Crohn's disease

NIH P01 DK 57880 (funded until 2010)  is a program project in which my lab investigates mechanisms of T and B cell adhesion and trafficking in experimental ileitis. A unique, spontaneous model of ileitis in mice, SAMP1/YitFc, develops lesions in the terminal ileum and responds to anti-inflammatory treatments. A closely related adoptive transfer model allows us to study T cell trafficking to these sites of inflammation. Recent discoveries include an important role for B cells in exacerbating the disease process. Also, we found that L-selectin-expressing T cells are, unexpectedly, involved in producing TNF-α, and blocking L-selectin in combination with another adhesion molecule, MAdCAM-1, ameliorates disease.

Olson, T.S., Bamias, G., Naganuma, M., Rivera-Nieves, J., Burcin, T.L., Ross, W., Morris, M.A, Pizarro, T.T., Ernst, P.B., Cominelli, F., Ley, K. (2004). Expanded B cell population blocks regulatory T cells and exacerbates ileitis a murine model of Crohn’s disease. J. Clin. Invest. 114: 389-398.

Rivera-Nieves J., Bruce A., Bamias G., Olson T., Burcin T., Hoang S., Pizarro T.T., Cominelli F., Ley K. (2005) L-selectin, α4b1 and α4b7 integrins regulate CD4+ T cell trafficking to chronically inflamed small intestine. Journal of Immunology,174: 2343-52.

Rivera-Nieves, J., Burcin, T., Olson, T.S., Morris, M.A., McDuffie, M., Cominelli, F., Ley, K. (2006). Critical role of endothelial PSGL-1 in chronic murine ileitis. J. Exp. Med. 203: 907-917

 

4. Molecular mechanisms of neutrophil recruitment to the lung

Project 2 of 1 P01 HL073361 (funded until 2009) is concerned with neutrophil trafficking to the normal and inflamed lung. We aim to 1) define the molecular mechanisms of neutrophil recruitment to the lung under resting and inflammatory conditions by studying neutrophil homing and measuring their accumulation in brochoalveolar lavage fluid (BAL), lung blood vessels, and in the interstitial spaces of the lung; 2) test whether endogenous chemokines are responsible for neutrophil recruitment by surveying chemokine expression by superarray, determining their localization by immunostaining or in situ hybridization where necessary, testing BAL for chemotactic activity and neutralizing candidate chemokines to gain mechanistic insights into the process; and 3) test the role of adenosine A2A receptors in regulating neutrophils recruitment in response to LPS, using A2A agonists, antagonists, A2A knockout mice, and chimeric mice generated by bone marrow transplantation.

Reutershan J, Basit A, Galkina E, Ley K.  (2005)  Sequential recruitment of neutrophils into lung and bronchoalveolar lavage fluid in LPS-induced acute lung injury.  Am.J. Physiol: Lung Cellular and Molecular Physiology  289: L807-15

Basit A, Reutershan J, Morris MA, Ley K.  (2006).  ICAM-1 and LFA-1 play critical roles in LPS-induced neutrophil recruitment into alveolar space. Am.J. Physiol: Lung Cell. Mol. Physiol. 291: L200-L207

Reutershan, J., Morris, M.A. Burcin, T.L. Smith, D.F., Chang, D. Saprito, M.S., Ley, K. (2006) Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung. J. Clin. Invest., 116:695-702

 

5. Neutrophil trafficking

Each day, more than 100 billion neutrophils are born in the bone marrow, circulate in the blood for a few hours and quietly do their job in peripheral tissues including the gut, lung, skin and mucosal membranes. The regulation of the processes associated with neutrophil trafficking are of great interest at all levels, from molecular to systemic. my lab explores neutrophil trafficking to the lung microvasculature and interstitium. Recently, we discovered a new class of T cells whose only purpose seems to be to regulate neutrophil production. We call them Tn cells for neutrophil regulatory T cells. This new class of T cells is critically involved in maintaining neutrophil homeostasis.

Stark, M.A., Burcin, T.L., Huo, Y., Morris, M.A., Olson, T.S., Ley, K. (2005) Phagocytosis of apoptotic neutrophils regulates granulopoiesis via IL-23 and IL-17. Immunity 22:  285-94.

Ley, K., Smith, E., Stark, M.A. (2006) Neutrophil-regulatory Tn lymphocytes. Immunologic Research 34: 229-242