Apoptosis and overexpression of Bax protein and bax mRNA in smooth muscle cells within intimal hyperplasia of human radial arteries - Analysis with arteriovenous fistulas used for hemodialysis

There is a type of arteriosclerosis with remodeling of middle-size arteries in which intimal hyperplasia of smooth muscle cells (SMCs) plays the main role, and there are few macrophages, T lymphocytes, and foam cells. It is u nknown whether apoptosis and the expression of Bar, an inducer of apoptosis , are increased according to the progression of this type of human arterios clerosis, which is different from so-called atherosclerosis, Bar heterodime rizes with Bcl-2, an inhibitor of apoptosis, and the ratio of Bar to Bcl-2 determines cellular apoptosis or survival. Thus, we investigated apoptosis and the expressions of Bax, bax mRNA, and Bcl-2 in human arteriovenous (AV fistulas used for hemodialysis, a representative of arteriosclerosis of the aforementioned type. The material was 20 radial arteries obtained from 20 patients with chronic renal failure undergoing AV shunt surgery. SMCs, macr ophages, and T lymphocytes were immunohistochemically identified at the lig ht microscopic (LM) level, Apoptosis was detected by in situ terminal deoxy nucleotidyl transferase (TdT)-mediated digoxigenin-dUTP nick end labeling ( TUNEL) at both the LM and electron microscopic (EM) level. Cell proliferati ng activity was estimated by proliferating cell nuclear antigen (PCNA), Bar and Bcl-2 were detected by immunohistochemistry and Western blot analysis. Expression of bax mRNA was detected by in situ hybridization, LM TUNEL-pos itive cells in both the intima and media were significantly increased accor ding to the percent stenosis of the vessels. EM analysis revealed that ultr astructures of apoptotic SMCs were seen in both synthetic and contractile p henotypes. Their frequency of occurrence in the intima and media were great er in those vessels with >50% stenosis than in those with <50% stenosis (5. 2+/-0.7% versus 1.0+/-0.3% in the intima and 2.1+/-0.5% versus 0.2+/-0.1% i n the media). The proportion of apoptotic SMCs with ruptured plasma membran es was greater than that of apoptotic SMCs with intact membranes in the int ima of the former (4.1+/-0.6% versus 1.1+/-0.1%). Only those SMCs with apop totic ultrastructures had TUNEL-positive nuclei with moderate or marked acc umulation of immunogold particles at the EM level. However, ultrastructures of oncosis (primary necrosis) were not observed. Immunohistochemical analy ses showed significant positive correlations between percent stenosis of ve ssels and the percentage of either PCNA-positive intimal cells or Bar-posit ive areas in the intima and media. Bcl-2-positive cells were not observed i n the intima but mainly in the outer media. The percentage of Bcl-2-positiv e medial cells was definitely decreased at an early stage after formation o f the AV fistula but did not change according to the duration of hemodialys is or the progression of arteriosclerosis, Western blot analysis of Bar or Bcl-2 and in situ hybridization of bax mRNA confirmed the immunohistochemic al data, Thus, regulation of cellularity in intimal hyperplasia of SMCs in human arteriosclerosis with remodeling is mediated by proliferation and apo ptosis but not oncosis. The apoptosis is probably induced by an increase in the Bar to Bcl-2 ratio.