Granzyme B in Atherosclerosis and Transplant Vascular Disease: Association with Cell Death and Atherosclerotic Disease Severity

Abstract

Apoptosis of intimal cells is an important contributor to the pathogenesis of atherosclerosis and transplant vascular disease (TVD).Since the activated immune response may be a key regulator of apoptosis in these lesions, we used immunohistochemistry to characterize the presence and localization of granzyme B, a major mediator of the cytotoxic immune response, in advanced atherosclerosis and TVD. Formalin-fixed, paraffin-embedded transverse sections from human left anterior descending coronary arteries were cut serially and stained with antibodies specific for granzyme B, smooth muscle ␣-actin, CD68, and CD3. The amount of granzyme B staining was semi-quantitated on a 0 -5؉/5؉ scale. Also, TUNEL staining and in situ hybridization was performed to visualize cells undergoing cellular damage suggestive of apoptosis, and to localize granzyme B mRNA, respectively. Granzyme B localization was similar in both diseases. This protease was absent in arteries with mild atherosclerosis, but was abundant in the intima and media of vessels with advanced atherosclerosis and TVD. Within the intima, granzyme B localized to TUNEL-positive foam cells surrounding lipid-rich atheromas. Staining of serial sections with granzyme B and either smooth muscle ␣-actin, anti-CD68, or anti-CD3 showed that granzyme B localized to smooth muscle cells, macrophages, and T-cells. Further, in situ hybridization for granzyme B mRNA in TVD cases localized its expression to infiltrating leukocytes and not foam cells. In conclusion, the presence of granzyme B in advanced atherosclerotic lesions and TVD is associated with increasing disease severity and cell death. These observations suggest that granzyme B-mediated apoptosis may contribute to the pathogenesis of these diseases.

Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin

“…37 In fact, atherosclerotic lesions in human coronary arteries have been shown to contain elevated numbers of mast cells 38 and an increased severity and cell death in advanced atherosclerotic lesions was associated with a presence of gzmB. 39 Furthermore, mast cell-derived gzmB may also contribute to vasculitis by inducing cell death in SMC, as reported. 11 However, only testing of atherosclerosis development in gzmB-deficient mice may provide evidence for the role of mast cell-derived gzmB in vascular diseases in vivo.…”

Perforin Mediates Endothelial Cell Death and Resultant Transplant Vascular Disease in Cardiac Allografts

“…10 We have also reported that granzyme B is abundant in TVD and that it localizes to apoptotic cells in these lesions. 11 Combined, these observations suggest that the perforin/granzyme pathway may be an important mediator of vascular damage in allograft vessels.…”

“…11 The number of TUNEL-positive cells in the endothelial region of coronary arteries from five hearts per group (n ϭ 16 arteries for each group) was counted manually and expressed as the average number of TUNEL-positive cells in the endothelial region/artery. TUNEL can also occasionally label necrotic cells as a result of uncontrolled DNA fragmentation that occurs during this form of cell death.…”

“…11 Briefly, sections were de-paraffinized and rehydrated in xylene and decreasing concentrations of ethanol. Antigen retrieval was performed by autoclaving slides in citrate buffer (pH 6.0) for 10 minutes.…”

Sinonasal T-Cell Expression of Cytotoxic Mediators Granzyme B and Perforin is Reduced in Patients with Chronic Rhinosinusitis

“…[34][35][36][37][38][39] Further investigation is required to determine the exact cell type that is responsible for the production of the remaining 10% of the cytotoxic mediators.…”