3-Deazaadenosine prevents smooth muscle cell proliferation and neointima formation by interfering with Ras signaling.

3-Deazaadenosine (c3Ado) is a potent inhibitor of S-adenosylhomocysteine hydrolase, which regulates cellular methyltransferase activity. In the present study, we sought to determine the effect of c3Ado on vascular smooth muscle cell (VSMC) function and neointima formation in vivo. c3Ado dose-dependently prevented the proliferation and migration of human coronary VSMCs in vitro. This was accompanied by an increased expression of the cyclin-dependent kinase inhibitors p21(WAF1/Cip1), p27(Kip1), a decreased expression of G(1)/S phase cyclins, and a lack of retinoblastoma protein hyperphosphorylation. In accordance with these findings, fluorescence-activated cell-sorting analysis of propidium iodide-stained cells indicated a cell cycle arrest in the G(0)/G(1) phase. Importantly, c3Ado did not affect the number of viable (trypan blue exclusion) or apoptotic cells (TUNEL). Mechanistically, c3Ado prevented FCS-induced Ras carboxyl methylation and membrane translocation and activity by inhibiting isoprenylcysteine carboxyl methyltransferase and reduced FCS-induced extracellular signal-regulated kinase (ERK)1/2 and Akt phosphorylation in a dose-dependent manner. Conversely, rescuing signal transduction by overexpression of a constitutive active Ras mutant abrogated c3Ado's effect on proliferation. For in vivo studies, the femoral artery of C57BL/6 mice was dilated and mice were fed a diet containing 150 microg of c3Ado per day. c3Ado prevented dilation-induced Ras activation, as well as ERK1/2 and Akt phosphorylation in vivo. At day 21, VSMC proliferation (proliferating-cell nuclear antigen [PCNA]-positive cells), as well as the neointima/media ratio (0.7+/-0.2 versus 1.6+/-0.4; P<0.05) were significantly reduced, without any changes in the number of apoptotic cells. Our data indicate that c3Ado interferes with Ras methylation and function and thereby with mitogenic activation of ERK1/2 and Akt, preventing VSMC cell cycle entry and proliferation and neointima formation in vivo. Thus, therapeutic inhibition of S-adenosylhomocysteine hydrolase by c3Ado may represent a save and effective novel approach to prevent vascular proliferative disease.

Correlation of vasa vasorum neovascularization and plaque progression in aortas of apolipoprotein E(-/-)/low-density lipoprotein(-/-) double knockout mice.

OBJECTIVE: We hypothesized that apolipoprotein E (apoE)(-/-)/low-density lipoprotein (LDL)(-/-) double knockout mice might develop vasa vasorum (VV) in association with advanced lesion formation. METHODS AND RESULTS: Aortas from apoE(-/-)/LDL(-/-) mice aged 16, 18, 20, or 80 weeks were infused in situ with Microfil, harvested, and scanned with micro-computed tomography (CT). We characterized plaque volume and CT "density" as well as VV luminal volume along the aorta using Analyze 6.0 software. Results were complemented by a detailed histological plaque classification according to American Heart Association guidelines. From 16 to 80 weeks, plaque volume and VV opacified lumen volume increased with age (P<0.001). The 3-dimensional micro-CT images of arterial and venous VV trees allowed perfusion territories to be delineated. The spatial location and magnitude of VV density and adventitial inflammation were strongly correlated in advanced atherosclerotic lesions (r=0.91) and identified as an independent correlate to advanced lesions. At age 80 weeks, VV luminal volume was increased 20-fold compared with animals at age 16 weeks (P<0.001). Micro-CT showed that adventitial VV communicate with intraplaque microvessels. CONCLUSIONS: Our results show that apoE(-/-)/LDL(-/-) double knockout mice develop VV and advanced atheromas along the aorta. Lesion volume was closely associated with amount of neovascularization in advanced atheromas.

Effects of 3-deazaadenosine on homocysteine and atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: In the past decade, elevated homocysteine concentration has achieved widespread recognition as an independent risk factor in the development of atherosclerosis. 3-Deazaadenosine (c3Ado) is a potent inhibitor and substrate for S-adenosylhomocysteine hydrolase and therefore may reduce homocysteine concentrations. The current study investigated the effect of c3Ado on serum homocysteine, atherosclerotic lesions, and the expression of adhesion molecules in apoE-knockout mice. METHODS AND RESULTS: Animals were placed on an atherogenic diet with or without c3Ado for 12 and 24 weeks. Frozen cross-sections of the aortic sinus and the proximal aorta were analyzed by computer-aided planimetry for fatty plaque formation. Macrophages, VCAM-1 and ICAM-1 were quantified by immunhistochemistry and oligo-cell reverse transcription polymerase chain reaction after laser microdissection. Application of c3Ado resulted in significant reduction of homocysteine levels by 35.9 and 45.3% after 12 and 24 weeks, respectively (P < 0.001). Neointimal area and atherosclerotic plaque formation were significantly reduced in animals treated with c3Ado (P < 0.01). Moreover, monocyte adhesion and concomitant ICAM-1 and VCAM-1 antigen and RNA expression on the endothelial layer were significantly reduced (P < 0.001, P < 0.01). CONCLUSION: Our results demonstrate that c3Ado induces a marked reduction of homocysteine concentrations which might explain in part the anti-atherogenic effect of the drug.

Protective effect of 3-deazaadenosine in a rat model of lipopolysaccharide-induced myocardial dysfunction.

Severe sepsis is accompanied by a profound depression of myocardial contractility. Leukocyte adhesion with subsequent local excess nitric oxide and reactive oxygen species production play major roles for this deleterious effect. We hypothesized that 3-deazaadenosine (c3Ado), an adenosine analogue with anti-inflammatory properties, prevents endotoxin-induced myocardial dysfunction. Wistar rats (8 per group) were treated with Escherichia coli lipopoly-saccharide (LPS, 1 mg/kg, i.p., strain 0111:B4) +/- c3Ado (10 mg/kg, i.p.) 8 h before their hearts were harvested for isolated perfusion, histochemical analysis, or electrophoretic mobility shift assay. LPS induced a marked depression of left ventricular contractility. Immunohistochemistry revealed an upregulation of the adhesion molecules VCAM-1, ICAM-1, and P-selectin within the postcapillary venules. c3Ado inhibited VCAM-1 and ICAM-1 upregulation, but not P-selectin, and prevented cardiodepression. Electrophoretic mobility shift assay revealed inactivation of the transcription factor nuclear factor-kappaB and immunohistochemical staining for gp91phox, ED1, and CD11b demonstrated that c3Ado prevented local recruitment of monocytes and polymorph nuclear neutrophils to the myocardium. Accordingly, significantly fewer leukocytes producing nitric oxide or reactive oxygen species accumulated within the myocardium. Intravital microscopy of intestinal venules confirmed that LPS-induced adhesion of leukocytes was prevented by c3Ado. Additionally, c3Ado prevented LPS-induced elevation of serum tumor necrosis factor-alpha levels. Our results imply that c3Ado may prove to have clinical relevance for inflammatory disease processes.

Atherosclerotic lesions at micro CT: feasibility for analysis of coronary artery wall in autopsy specimens.

PURPOSE: To evaluate the feasibility of micro computed tomography (CT) for analysis of the coronary artery wall. MATERIALS AND METHODS: With micro CT, two-dimensional transverse images were generated from 10 human autopsy specimens of coronary arteries (2.5-3.5 cm long), with section thickness of 6 microm. Vessel wall perimeter, plaque area, calcified lesion area, media area, and lumen area were determined by three experienced radiologists. Results were compared with those obtained from a detailed conventional histomorphometric analysis of corresponding cross sections. Hotelling T(2) test (a multivariate generalization of the univariate Student t test) and Pearson correlation coefficient were used to assess the correlation between micro CT findings and conventional histologic measurements. The significance of differences in gray-scale measurements was tested with analysis of variance. RESULTS: Micro CT provided quantitative information about plaque morphology equivalent to that provided with histomorphometric analysis. Hotelling T(2) test revealed significantly smaller values for vessel wall perimeter and lumen area with histologic sections (P <.001). Gray-scale measurements were established with which lesions could be categorized after histologic classification. CONCLUSION: Micro CT is feasible for analysis of the coronary artery wall.