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Accelerated intimal thickening in carotid arteries of balloon-injured rats after immunization against heat shock protein 70

Jacob George, MD^*, Shai Greenberg, MSc^*, Iris Barshack, MD, Madhavir Singh, PhD, Sara Pri-Chen, PhD^*, Shlomo Laniado, MD^* and Gad Keren, MD^*,*

^* Department of Cardiology and the Cardiovascular Research Laboratory, Tel Aviv Medical Center, Tel Aviv, Israel
Institute of Pathology, Sheba Medical Center, Tel Hashomer, Israel
GBF-Braunschweig, Braunschweig, Germany

View larger version (14K): [in a new window]   Figure 1 Kinetics of anti-heat shock protein (Hsp) 70 antibody production. Rats immunized with Hsp70, bovine serum albumin (BSA) or control were bled before (t = 0), 10 days after primary immunization (t = 1), at the time of injury (t = 2) or at sacrifice (t = 3). Anti-Hsp70 antibody levels were determined by enzyme-linked immunosorbent assay. Values represent mean ± SEM of OD from all animals in each group. Upward-pointing solid triangle = BSA; downward-pointing solid triangle = control; solid circle = Hsp70.

View larger version (40K): [in a new window]   Figure 2 Time dependence of heat shock protein (Hsp) 70 expression in injured rat carotid arteries. Injured or noninjured carotid arteries were harvested from animals at different time points before and after balloon injury. Heat shock protein 70 expression was determined by Western blot combined with enhanced chemiluminescence, employing polyclonal mouse anti-Hsp70 (A). Representative samples taken 3 and 14 days after arterial injury are shown. Densitometric analysis of reactivity with Hsp70 of injured arteries (average of three vessel per bar) from panel A (B).

View larger version (101K): [in a new window]   Figure 3 Immunohistochemical detection of heat shock protein (Hsp) 70 in rat carotid arteries. Frozen sections of injured (A) and noninjured (B) carotid arteries were frozen and embedded in OCT. Immunohistochemical detection of expressed Hsp70 was carried out employing mouse anti-Hsp70 polyclonal antibody as a primary antibody. The arrow indicates neointima-media border.

View larger version (11K): [in a new window]   Figure 4 Intimal thickening in rats immunized with heat shock protein (Hsp) 70. Rats were immunized and boosted with Hsp70, bovine serum albumin (BSA) or phosphate-buffered saline (PBS) (control). Two weeks after boost, balloon injury was applied to carotid arteries of both groups. Morphometric evaluation of intimal (A) and medial (B) area were performed using PhotoShop software. The ratio of intima to medial area was also calculated (C). Values represent mean ± SD of all animals in each group. *p = 0.001 as compared with control.

View larger version (129K): [in a new window]   Figure 5 Representative injured carotid arteries immunized with heat shock protein (Hsp) 70 or control. Representative hematoxylin & eosin staining of carotid arteries from Hsp70-immunized rats (A) as compared with control-injected rats (B) two weeks after balloon injury. Original magnification x25.

View larger version (28K): [in a new window]   Figure 6 Heat shock protein (Hsp) 70 reactivity of immunoglobulin G fractions eluted from injured and noninjured carotid arteries of Hsp70 immunized rats. Immunoglobulin G eluted from carotid arteries of injured (striped bar) and noninjured (black bar) Hsp70-immunized animals were characterized by assessing their binding to solid phase coated Hsp70 or an irrelevant antigen (bovine serum albumin). Values represent mean ± SEM of three tissue samples in each group.