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Heat shock proteins (HSPs) of endogenous and exogenous origin are suspected contributors to the initiation and aggravation of vascular pathologies like atherosclerosis and restenosis. Toll-like receptors (TLRs) 2 and 4 are well-known receptors for exogenous pathogen-associated molecular patterns and have recently been thought to play a role in HSP60-induced cellular activation. We hypothesized that human HSP60 directly stimulates venous smooth muscle cell (VSMC) proliferation through a TLR-dependent mechanism. Localization of HSP60, TLR2 and TLR4 was studied in failed venous grafts and normal venous tissue by double immunostaining. In vitro VSMCs were incubated for 48 h with recombinant human HSP60. In other experiments, VSMCs were pre-incubated for 30 min with specific anti-TLR2 and anti-TLR4 antibodies. VSMC proliferation was determined by Ki67 immunoreactivity, and mean values were compared between experimental and control groups. In addition, human embryonic kidney (HEK) cells transfected with human TLR2 or TLR4/MD-2 were exposed to HSP60 for 48 h, and proliferation was determined by using a hemocytometer. Co-localization of HSP60 and TLRs was detected in all neointimal lesions but was virtually absent in normal veins. Human HSP60 stimulated VSMC proliferation in a concentration-dependent fashion. In addition, TLR2 and TLR4 antibodies attenuated VSMC proliferation. The role of TLR-mediated stimulation of cell proliferation by HSP60 was supported by the significant increase in proliferation of transfected HEK cells. These findings provide supporting evidence for the role of HSP60 and TLR2 and TLR4 in vascular disease. Moreover, our data surpass the infection- and autoimmunity-based hypotheses of cardiovascular disease and suggest an additional HSP60-related autocrine process.

Epidemiological and animal studies suggest a role for cytomegalovirus (CMV) in restenosis. Previously, we demonstrated that proliferating smooth muscle cells (SMCs) in the injured arterial wall are particularly susceptible to CMV-induced effects. Therefore, we hypothesised that, depending on the time point of infection after vascular injury, CMV infection may affect cell proliferation either in the media or in the neointima, thereby aggravating the process of restenosis. In the present study, we focused on the individual layers of the arterial wall by evaluating, besides the neointima-to-media ratio, the medial and neointimal area and cellularity in the rat femoral artery. Vascular injury was photochemically induced in rat femoral arteries. Immediately or 14 days thereafter, rats were infected with rat CMV (RCMV) or mock infected. The presence of RCMV in the vascular wall was determined at 3, 5, 14 and 35 days after infection by quantitative real-time PCR. When rats were infected immediately after injury, a significant increase was seen only in the medial but not in the neointimal cross-sectional area. On the other hand, when rats were infected 14 days after the initial injury, a significant increase was only seen in the neointimal area, thereby confirming our hypothesis that RCMV infection primary affects proliferating SMCs. As the mean number of SMCs per microm2 in both cell layers was unchanged despite an increase in cross-sectional area, this implies that RCMV stimulated SMC proliferation. Furthermore, these vascular effects were observed without the virus being abundantly present in the vascular wall, suggesting that inflammatory and immune-mediated responses to RCMV infection are more important in aggravating the response to vascular injury than the virus itself.

We have previously demonstrated that mouse cytomegalovirus (MCMV) aggravates atherosclerosis in apolipoprotein E knockout (apoE(-/-)) mice, most likely by enhancing both systemic and local (e.g. in the vascular wall) cytokine production. However, until now it was unclear which cell type is responsible for this enhanced pro-inflammatory cytokine production. In this study we focused on the macrophage (mPhi), which besides being an important source of such cytokines, is known to be an important player in both atherosclerosis and viral clearance. We investigated whether MCMV could induce a pro-inflammatory immune mPhi phenotype, which ultimately may contribute to the development of atherosclerosis. To this end, peritoneal exudate cells (PEC) were elicited in apoE(-/-) mice by either MCMV or thioglycolate injection, and mPhi were phenotyped at 1 week post-intraperitoneal injection. MCMV-induced peritoneal mPhi contained MCMV DNA but had limited MCMV mRNA expression, indicating latent infection. These mPhi showed increased production of interferon-gamma (IFNgamma), exclusive production of interleukin-18 (IL-18) and increased expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86, when compared with thioglycolate-induced mPhi. From these results, we conclude that intraperitoneal injection of MCMV induces an immune-responsive exudate in which at 7 days post-infection, MCMV-infected mPhi express a pro-inflammatory immune phenotype. As such, the MCMV-induced mPhi may be an important player in aggravating atherosclerosis through systemic and/or local immune activation.

Since the 1970s, cytomegalovirus (CMV) infection has been associated with atherosclerotic disease. However, the exact contribution of the virus remains uncertain. In this article we describe both a direct and indirect immune-mediated effect of the virus on the disease process. Eight-week-old apolipoprotein E (apoE) knockout mice were infected with mouse CMV (MCMV) or mock injected, and they were sacrificed at 2 and 20 weeks post-injection (p.i.) to study atherosclerosis, vascular wall IFNgamma and TNFalpha expression and MCMV spread. To study plasma IFNgamma and TNFalpha levels, blood was collected at 1, 2, 4 and 6 days p.i. in addition to days of sacrifice. Plasma cytokine levels were increased after MCMV infection at early time points and decreased to mock levels at 2 and 20 weeks p.i. At 2 weeks p.i., more aortic arch samples showed local cytokine expression after MCMV infection. The number of early atherosclerotic lesions and the percentage of mice containing early lesions were increased at 2 weeks p.i., while at 20 weeks p.i., the MCMV-induced effect on atherogenesis was seen on the late lesions. In conclusion, MCMV infection induces a systemic immune response reflecting an indirect effect of MCMV infection on atherosclerosis in addition to a local aortic immune response reflecting a direct effect of the virus on the atherosclerotic process.

The mono- and di-iodinated analogs of lysine and arginine vasopressin have been prepared previously but not well characterized chemically. Their biological activities are greatly reduced with respect to LVP or AVP. In this paper we report a convenient synthesis of iodinated AVP agonists and antagonists, their purification by high performance liquid chromatography, and their characterization by fast atom bombardment mass spectrometry. In contrast to the results obtained with agonists, the mono-iodinated vasopressin antagonist retains virtually the full receptor activity of its non-iodinated parent. This should allow the preparation of labeled vasopressin antagonists of high specific activity for receptor characterization and isolation.