Inhibitory effect on arterial injury-induced neointimal formation by adoptive B cell transfer in Rag-1 KO mice

P. Dimayuga, B. Cercek, S. Oguchi, G. N. Fredrikson,J. Yano, P. K. Shah, S. Jovinge, J. Nilsson

Department of Medicine, Lund University, University Hospital MAS, Malmö, Sweden Atherosclerosis Research Center, Cedars-Sinai Medical Center, Los Angeles, California;

Department of Biomedical Laboratory Science, Health and Society, Malmö University, Malmö, Sweden

General Findings:

• Immune-deficiency results in increased neointimal formation after arterial injury in mice.

• Humoral aspects of the immune system may be involved in inhibiting neointimal formation:

B cells produce “natural antibodies” ?

Background•T cell deficiency results in increased neointima in injured rats (antibodies against T cell CD markers and nu/nu rats)

Hansson, et al PNAS/Circ 1991 Remskar et al Circ Res. 2001

•CD40-CD40 ligand interaction mediates response to arterial injury

Remskar et al Circ Res. 2001

•B cells implicated in bone-healing response as a limiting factor

Marusic, et al LI 2000

•Homologous oxLDL immunization reduces neointimal formation in injured hypercholesterolemic rabbits

Nilsson et al JACC 1997

Arterial Injury

Activation of signaling cascades

Increased expression of inflammatory proteins/medial SMC activation

Immune functions physiologic IFN- Immunoglobulins

Immune-deficiency

Vascular repair and Intimal formation

Hypothesis: B cells are involved in the response toarterial injury.

Methods:•Injure Rag-1 KO mice and compare to WT mice.•“Rescue” Rag-1 KO mice with B cell reconstitutionfrom spleens of WT•Repeat rescue with donor and recipients on westerndiet

Arterial Cuff Injury

Tygon tube

Right carotid artery

harvest spleen from WT mice

squeeze through mesh to isolate cells

add paramagnetic beadscoated with T cell antibody

expose solution to magnetic field andaspirate supe = negative selection of B cells

classify cell populationand/or spin and concentrate

Inject cells into Rag-1 KO mice via tail vein

Cell enrichment procedure

*p<0.05 vs. WT NC; †p<0.05 vs. Rag NC

B cell reconstitution inhibits intimal formationin Rag-1 KO mice fed normal chow

B cell reconstitution inhibits intimal formationin Rag-1 KO mice fed western diet

*p<0.05 vs. WT HC; †p<0.05 vs. Rag HC

WT HC Rag-KO Rag-1 KO Brec

Effect of B cell reconstitution onintimal formation in injured Rag-1 KO mice

Neointimal stain for SM -actin(western diet-fed mice)

WT Rag-1 KO Rag-1 KO Brec

M stain in arteries 21 days after injury(western diet-fed mice)

WT Rag-1 KO Rag-1 KO Brec

IgM in injured arteries of western diet-fed mice

Left contra-lateral (uninjured) Injured right carotid artery

No reconstitution B cell reconstituted

WT

Rag-1 KO

WT

Rag-1 KO

Left contra-lateral (uninjured) Injured right carotid artery

No reconstitution B cell reconstituted

IgG in injured arteries of western diet-fed mice

Relative immunoglobulin levelsin B cell reconstituted mice

Summary of Study

•Neointimal formation after injury is inhibited in Rag-1KO mice reconstituted with B cells regardless of diet.

•Immunoglobulin presence is increased in injuredarteries of WT mice and in B cell reconstituted Rag-1KO mice.