Allan Sirsjö

Differential expression of cysteine and aspartic proteases during progression of atherosclerosis in apolipoprotein E-deficient mice

Several groups of proteolytic enzymes are able to degrade components of the extracellular matrix. During atherosclerosis, matrix remodeling is believed to influence the migration and proliferation of cells within the plaque. In the present study, gene expression of several proteases and their inhibitors was analyzed during the development of atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. Quantitative real-time polymerase chain reaction was used to study gene expression of proteases after 10 and 20 weeks in ApoE-/- and C57BL/6 mice and in atherosclerotic lesions and nonaffected regions of the same ApoE-/- mouse. Some of the differentially expressed proteolytic enzymes were studied by immunohistochemistry. The matrix metalloproteinase (MMP)-9 and its inhibitor TIMP-1 were differentially expressed and the expression increased with time. Urokinase-type plasminogen activator showed no major expression. In contrast, cathepsins B, D, L, and S all showed strong and increased expression in ApoE-/- mice compared to C57BL/6 mice whereas the expression of their inhibitor, cystatin C, did not differ between the two mouse strains. The expression of cathepsins was mainly localized to the lesions and not to nonaffected regions of the aorta of ApoE-/- mice. Furthermore, cathepsin expression was similar to the expression of the macrophage marker macrosialin (CD68) although expression of cathepsins B, D, and L could be demonstrated in healthy C57BL/6 mice and in nonaffected vessel segments of atherosclerotic ApoE-/- mice. Cathepsin S mRNA expression was restricted to lesions of ApoE-/- mice. Furthermore, cathepsin S was the only cathepsin that was expressed in the media and absent in lipid-rich regions. All cathepsins studied showed intimal expression, the degree and localization of which differed between individual cathepsins. In conclusion, increased expression of several cathepsins in atherosclerotic lesions suggests that these proteases may participate in the remodeling of extracellular matrix associated with the atherosclerotic process.

Retinoic Acid Regulates Arterial Smooth Muscle Cell Proliferation and Phenotypic Features In Vivo and In Vitro Through an RARα-Dependent Signaling Pathway

We have recently shown that all-trans retinoic acid (tRA) modulates arterial smooth muscle cell (SMC) morphologic features and biochemical composition in vitro. It has been proposed that different SMC phenotypes coexist in arteries, which may be retrieved in culture: hence, a differential action of tRA on distinct SMC subsets is conceivable. We have examined the effect of tRA on SMC proliferation, migration, plasminogen activator activity, and alpha-smooth muscle actin expression in 2 phenotypically different rat SMC populations, cultured respectively from the normal aortic media and from the intimal thickening (IT) after endothelial injury. tRA inhibited proliferation and increased migration and tissue-type plasminogen activator activity in both SMC populations, but decreased alpha-smooth muscle actin only in SMC cultured from the IT. The action of tRA is mediated by 2 families of nuclear receptors, RAR and RXR, each containing 3 isoforms, alpha, beta, and gamma. RAR and RAR-alpha agonists, but not RXR agonists, inhibited SMC proliferation in both cell populations and alpha-smooth muscle actin expression only in IT SMC. When administered intraperitoneally to balloon-injured rats, tRA and RAR-alpha agonists reduced the intimal hyperplasia in the carotid artery. Our results show that tRA and synthetic retinoids can affect the proliferation, migration, and differentiation of SMC in vitro. Furthermore, retinoids are able to reduce the IT induced by endothelial injury in vivo.

Expression of interleukin-15 in mouse and human atherosclerotic lesions

Atherosclerotic lesions are characterized by prominent macrophage and T-cell infiltration and atherosclerosis is widely recognized as an inflammatory disease. The cytokine interleukin-15 (IL-15) has T-cell chemotactic and pro-inflammatory properties and promotes the recruitment of T cells to sites of inflammation. We have therefore examined IL-15 expression in the atherosclerotic ApoE-deficient mouse model as well as in human atherosclerotic lesions. In gene expression arrays, a transcript corresponding to IL-15 mRNA was elevated in atherosclerotic aortas of ApoE-deficient mice fed a Western diet for 10 and 20 weeks, corresponding to lesions of the fatty streak and fibrofatty plaque stage, respectively. Immunostaining for IL-15 localized to aortic smooth muscle cells in nonatherosclerotic C57BL/6 mice, whereas both macrophages and smooth muscle cells stained positive for IL-15 in atherosclerotic lesions of ApoE-deficient mice. Finally, advanced atherosclerotic lesions of human carotid arteries were immunostained to determine whether IL-15 is involved in human disease. IL-15 protein was present also in the human lesions with a distribution primarily overlapping that of macrophages. In conclusion, IL-15 is up-regulated in both human and animal atherosclerotic lesions and may contribute to the recruitment of T cells and their activation during atherogenesis.

The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque

BACKGROUND Although the association between inflammation and atherosclerosis is well established, the biologic events that trigger the local inflammatory response within plaque are not fully understood. Cytotoxic free radicals and infectious agents, both of which are associated with an inflammatory response, have previously been implicated in the initiation and progression of atherosclerosis. In this study, we analyzed carotid plaque for evidence of oxidative vascular injury by determining the presence and distribution of inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation and for evidence of infection with cytomegalovirus. METHODS Carotid plaque from 51 patients who underwent endarterectomy for either primary (n = 37) or recurrent (n = 14) stenosis were examined histologically (hematoxylin-eosin staining and Massons trichrome staining) and with immunohistochemistry with specific antibodies to alpha-smooth muscle actin, macrophages (CD68), T-lymphocytes (CD3), and T-cell activation (human leukocyte antigen-DR). Twenty-eight specimens from patients with primary (n = 15) and recurrent (n = 13) stenosis were examined for the presence of iNOS and nitrotyrosine with immunohistochemistry and in situ hybridization (iNOS). Twenty-three additional specimens (22 primary, and 1 recurrent) were analyzed with antibodies to p53, cytomegalovirus, and the polymerase chain reaction (cytomegalovirus, n = 8). RESULTS Primary atherosclerotic lesions were either complex heterogenous cellular plaques (n = 29) or relatively acellular fibrous plaques (n = 8). Ten of 14 recurrent plaques were either complex or fibrous lesions, and the remaining four were typical of myointimal thickening. CD68-positive staining cells were detected in all specimens regardless of their structural morphology. CD3-positive cells were interspersed between macrophages in all heterogeneous cellular plaques and only infrequently noted in fibrous plaques. iNOS and nitrotyrosine immunoreactivity were detected in macrophages and smooth muscle cells in all complex and fibrous plaques and in two of four myointimal plaques. The presence of iNOS and nitrotyrosine in plaque correlated with the existence of symptoms in 80% of primary and 62% of recurrent lesions. Cytomegalovirus was detected in only two of 23 carotid specimens (9%). CONCLUSION The association between ischemic cerebrovascular symptoms and iNOS and nitrotyrosine immunoreactivity in complex primary and recurrent carotid plaque and the infrequent occurrence of cytomegalovirus in primary carotid lesions suggests that ongoing free radical oxidative damage rather than viral infection may contribute to plaque instability in patients with complex and fibrous carotid plaques.