Selective inactivation of p27(Kip1) in hematopoietic progenitor cells increases neointimal macrophage proliferation and accelerates atherosclerosis

Excessive proliferation of immune cells and vascular smooth myocytes (VSMCs) contributes to atherosclerosis. We have previously shown that whole-body inactivation of the growth suppressor p27 exacerbates atherosclerosis in apolipoprotein E-null mice (apoE-/-), and this correlated with increased proliferation of arterial macrophages and VSMCs. In the present study, we postulated that targeted disruption of bone marrow (BM) p27 is sufficient to enhance arterial macrophage proliferation and atherosclerosis. To test this hypothesis, sublethally irradiated apoE-/- mice with an intact p27 gene received a BM transplant from either apoE-/- or p27-/-apoE-/- doubly deficient donor mice and challenged with a high-cholesterol diet. Compared with mice that received an apoE-/- BM transplant, reconstitution with p27-/-apoE-/- doubly deficient marrow increased the expression of proliferating cell nuclear antigen in neointimal macrophages and accelerated aortic atherosclerosis, and this correlated with augmented aortic expression of the inflammatory cytokines CCL2/MCP-1 (monocyte chemoattractant protein 1) and CCL5/RANTES (regulated on activation, normal T-cell expressed and secreted). Overall, these findings provide evidence that p27 deficiency in hematopoietic progenitor cells enhances the inflammatory/proliferative response induced by dietary cholesterol and accelerates atherosclerosis.     

Angiotensin II infusion induces site-specific intra-laminar hemorrhage in macrophage colony-stimulating factor-deficient mice

Angiotensin II (AngII) infusion promotes macrophage infiltration into the aortic wall resulting in several forms of vascular pathology. To determine the causal role of macrophages in these vascular diseases, we used osteopetrotic (op) male mice in which a natural mutation ablates production of M-CSF and results in severe depletion of monocytes. AngII infusion into apoE-/- mice resulted in increased atherosclerosis that was attenuated in op mice. AngII infusion in op mice unexpectedly produced grossly discernable thickening of the proximal thoracic aorta characterized by intra-mural hematoma. This pathology was also observed in apoE+/+ x op male mice, and therefore, independent of hyper-lipidemia. No perceptible structural properties of aortas from op mice could be discerned prior to AngII infusion. Regional effects in the contractile response to phenylephrine were noted in aortic rings with enhanced responsivity in the upper thoracic aortas of op mice compared to those from +/+ mice. No differences in contractile response were noted in aortic rings from the lower thorax. In conclusion, deficiency of M-CSF attenuated AngII-induced atherosclerosis but led to an unanticipated pathology of intra-laminar hemorrhage in the upper aortic regions.     

Atherosclerosis progression in LDL receptor-deficient and apolipoprotein E-deficient mice is independent of genetic alterations in plasminogen activator inhibitor-1

Impaired fibrinolysis has been linked to atherosclerosis in a number of experimental and clinical studies. Plasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of plasminogen activation and has been proposed to promote atherosclerosis by facilitating fibrin deposition within developing lesions. We examined the contribution of PAI-1 to disease progression in 2 established mouse models of atherosclerosis. Mice lacking apolipoprotein E (apoE-/-) and mice lacking the low density lipoprotein receptor (LDLR-/-) were crossbred with transgenic mice overexpressing PAI-1 (resulting in PAI-1 Tg(+)/apoE-/- and PAI-1 Tg(+)/LDLR-/-, respectively) or were crossbred with mice completely deficient in PAI-1 gene expression (resulting in PAI-1-/-/apoE-/- and PAI-1-/-/LDLR-/-, respectively). All animals were placed on a western diet (21% fat and 0.15% cholesterol) at 4 weeks of age and analyzed for the extent of atherosclerosis after an additional 6, 15, or 30 weeks. Intimal and medial areas were determined by computer-assisted morphometric analysis of standardized microscopic sections from the base of the aorta. Atherosclerotic lesions were also characterized by histochemical analyses with the use of markers for smooth muscle cells, macrophages, and fibrin deposition. Typical atherosclerotic lesions were observed in all experimental animals, with greater severity at the later time points and generally more extensive lesions in apoE-/- than in comparable LDLR-/- mice. No significant differences in lesion size or histological appearance were observed among PAI-1-/-, PAI-1 Tg(+), or PAI-1 wild-type mice at any of the time points on either the apoE-/- or LDLR-/- genetic background. We conclude that genetic modification of PAI-1 expression does not significantly alter the progression of atherosclerosis in either of these well-established mouse models. These results suggest that fibrinolytic balance (as well as the potential contribution of PAI-1 to the regulation of cell migration) plays only a limited role in the pathogenesis of the simple atherosclerotic lesions observed in the mouse.     

Site-specific antiatherogenic effect of probucol in apolipoprotein E-deficient mice

-The lipid-lowering antioxidant probucol can inhibit atherosclerosis in animals and restenosis in humans. However, probucol has been shown to promote atherosclerosis in the aortic root of apolipoprotein E-deficient (apoE-/-) mice. In the current study, we examined the effects of probucol on both lesion formation at 4 sites along the aorta and lipoprotein oxidation in the plasma and aortas of apoE-/- mice receiving a diet containing 21.2% (wt/wt) fat and 0. 15% (wt/wt) cholesterol without or with 1% (wt/wt) probucol. After 6 months, controls had developed lesions at all sites investigated. Lesion development was strongly (P=0.0001) affected by probucol, but this effect was not uniform: lesion size was increased in the aortic root but significantly decreased in the arch, the descending thoracic aorta, and proximal abdominal aorta. Plasma and aortas of probucol-treated mice contained high concentrations of probucol and its metabolites (bisphenol and diphenoquinone); increased vitamin C; markedly decreased very low density lipoprotein (but not low density lipoprotein and high density lipoprotein); and decreased cholesterol, cholesteryl esters, triglycerides, vitamin E, and oxidized lipids compared with controls. Interestingly, probucol treatment did not decrease the proportion of aortic lipids that were oxidized. Plasma vitamin C and bisphenol, but not probucol, protected plasma lipids from ex vivo oxidation by peroxyl radicals. These results show that as in other species, probucol can inhibit lesion formation in most parts of the aorta of apoE-/- mice. This effect may involve lipid oxidation-independent mechanisms localized within the vessel wall as well as lipid lowering.     

Influence of increased age on the development of herpes stromal keratitis

Herpes stromal keratitis (HSK) is the leading infectious cause of blindness in the United States and is a consequence of events following HSV-1 infection of the eye. The pathology of the disease is currently thought to be caused by a destructive, CD4(+) T helper 1 (Th1) type inflammatory immune response within the cornea rather than a cytopathic response elicited by the virus. A large percentage of people can become infected with HSV-1 as children whereas some studies have concluded that many others do not become infected with HSV-1 until much later in life. In this paper we investigate the role of increasing age on ocular HSV-1 infection. Following an ocular infection of mice with HSV-1 we observed greater pathology in the cornea during both early and late time-points in adult mice when compared to young animals. No significant differences in viral titers were observed in either the eyes or trigeminal ganglia from infected mice, regardless of age, suggesting that increased viral load may not be responsible for the ocular pathology in the adult mice. We hypothesize that age-related changes in the immune response may predispose adult animals to HSK disease.     

Myocarditis in newborn wild-type BALB/c mice infected with the murine gamma herpesvirus MHV-68

OBJECTIVES: Animal models of human Epstein-Barr virus (EBV) infection include EBV infection of primates and infection of mice with MHV-68, a further gamma herpesvirus (gamma-HV). We aimed at extending the MHV-68 model to study gamma-HV-related cardiac disease. METHODS: Newborn wild-type BALB/c- (n=107), wild-type C57BL/6- (n=17) and immunodeficient B6-(Rag1) mice (n=18) were infected by nasal inoculation and evaluated for histopathological changes as well as tissue viral loads. RESULTS: From day 5 on BALB/c mice showed myocardial viral replication. Whereas focal inflammation occurred simultaneously, necrosis was first observed 9 days post-infection. The maximum rates of necrosis (40%) and of focal inflammation (33%) were found after 10 to 12 and 33 to 35 days, respectively. Some animals developed persistent viral activity and inflammation throughout the observation period of three months. Inflammation was mainly related to T cell infiltrates. Although C57BL/6 mice also showed myocardial inflammation, necrosis was not found suggesting differences in the susceptibility to the virus in distinct mouse strains. In immunodeficient animals higher myocardial viral loads were observed compared to wild-type mice but no cardiac lesions, which suggests that the antiviral immune response contributed to the lesions. CONCLUSIONS: The model system presented here is the first to allow detailed studies on cardiac disease caused by gamma-HV infections and may facilitate the development of more specific treatment options for human cardiac EBV infection.     

Accumulation of biglycan and perlecan, but not versican, in lesions of murine models of atherosclerosis

Proteoglycan accumulation within the arterial intima has been implicated in lipoprotein retention and in atherosclerosis progression in humans. Two commonly studied murine models of atherosclerosis, the apolipoprotein E (apoE)-deficient (apoE-/-) mouse and the low density lipoprotein receptor-deficient (LDLR-/-) mouse, develop arterial lesions similar to those of human atherosclerosis. However, specific proteoglycan classes that accumulate in lesions of these mice and their relation to the retention of specific apolipoproteins have not been previously determined. In this report, we characterized the distribution of proteoglycans (versican, biglycan, and perlecan) and apolipoproteins (apoB, apoA-I, and apoE) in proximal aortic lesions of chow-fed apoE-/- and LDLR-/- mice at 10, 52, and 73 weeks of age. We observed that similar to the apoE-/- mice, the LDLR-/- mice develop intermediate and advanced plaques within 52 weeks of age. Perlecan and biglycan (both are proteoglycans) appeared early in lesion development with distinct expression patterns as the plaques advanced. Versican, a major proteoglycan detected in human plaques, was mostly absent in both strains. ApoA-I and apoB were detected in early through advanced lesions in regions of proteoglycan accumulation in both strains. Our results indicate that proteoglycans may contribute to the retention of lipoproteins at the earliest stage of atherosclerosis in murine models of atherosclerosis.     

The potential role of resistin in atherogenesis

Resistin, an adipocyte-derived cytokine linked to insulin resistance and obesity, has recently been shown to activate endothelial cells (ECs). Using microarrays, we found that along with numerous other pro-atherosclerotic genes, resistin expression levels are elevated in the aortas of C57BL/6J apoE-/- mice; these findings led us to further explore the relation between resistin and atherosclerosis. Using TaqMan PCR and immunohistochemistry, we found that ApoE-/- mice had significantly higher resistin mRNA and protein levels in their aortas, and elevated serum resistin levels, compared to C57BL/6J wild-type mice. Incubation of murine aortic ECs with recombinant resistin increased monocyte chemoattractant protein (MCP)-1 and soluble vascular cell adhesion molecule (sVCAM)-1 protein levels in the conditioned medium. Furthermore, human carotid endarterectomy samples stained positive for resistin protein, while internal mammary artery did not show strong staining. Patients diagnosed with premature coronary artery disease (PCAD) were found to have higher serum levels of resistin than normal controls. In summary, resistin protein is present in both murine and human atherosclerotic lesions, and mRNA levels progressively increase in the aortas of mice developing atherosclerosis. Resistin induces increases in MCP-1 and sVCAM-1 expression in murine vascular endothelial cells, suggesting a possible mechanism by which resistin might contribute to atherogenesis. Finally, PCAD patients exhibited increased serum levels of resistin when compared to controls. These findings suggest a possible role of resistin in cardiovascular disease.     

Gammaherpesvirus-Induced Lung Pathology Is Altered in the Absence of Macrophages

The purpose of this study was to examine the lung pathogenesis of murine gammaherpesvirus (MHV-68) infection in mice that lack CC chemokine receptor CCR2, an important receptor for macrophage recruitment to sites of inflammation. BALB/c and CCR2^−/− mice were inoculated intranasally (i.n.) with MHV-68 and samples were collected during acute infection (6 dpi) and following viral clearance (12 dpi). Immunohistochemistry was used to determine which cells types responded to MHV-68 infection in the lungs. Lung pathology in infected BALB/c mice was characterized by a mixed inflammatory cell infiltrate, necrosis, and increased alveolar macrophages by 12 dpi. Immunohistochemistry showed intense positive staining for macrophages. CCR2^−/− mice showed greater inflammation in the lungs at 12 dpi than did BALB/c mice, with more necrosis and diffuse neutrophil infiltrates in the alveoli. Immunohistochemistry demonstrated much less macrophage infiltration in the CCR2^−/− mice than in the BALB/c mice. These studies show that CCR2 is involved in macrophage recruitment in response to MHV-68 infection and illustrates how impairments in macrophage function affect the normal inflammatory response to this viral infection.     

Tumor necrosis factor-alpha and interleukin-1 beta play a critical role in the resistance against lethal herpes simplex virus encephalitis

BACKGROUND: The innate immune response after herpes simplex type 1 (HSV-1) encephalitis could be protective or, paradoxically, implicated in neuronal damage. We investigated the role of the innate immune response in such infection using a C57BL/6 mouse knockout (KO) model for tumor necrosis factor (TNF)-alpha and/or interleukin (IL)-1beta. METHODS: Encephalitis was induced by intranasal infection with a clinical strain of HSV-1 in 1-month-old KO or wild-type (WT) mice. Mice were monitored for survival, brain viral load was quantified by real-time polymerase chain reaction, and the inflammatory response was assessed by in situ hybridization in groups of mice killed on days 3-7. RESULTS: WT mice had a significantly higher mean life expectancy (P=.0001, log-rank test) than other groups. IL-1beta and TNF-alpha KO mice had a similar mean life expectancy, and encephalitis was lethal to all TNF-alpha /IL-1beta-deficient mice. Brain viral loads were lower in WT than in KO mice that had disseminated viral replication in the pons and medulla. Moreover, TNF- alpha and IL-1beta KO mice failed to initiate an adequate immune response, as shown by the virtual absence of expression of proinflammatory molecules in the brain. CONCLUSION: These data clearly demonstrate the importance of TNF-alpha and IL-1beta in protection against HSV-1 encephalitis in this mouse model.     

Evaluation of vascular function in apolipoprotein E knockout mice with angiotensin-dependent renovascular hypertension

It is known that the endothelial function is compromised in atherosclerosis and arterial hypertension and that angiotensin is an important factor contributing to both pathophysiologies. The aim of this study was to evaluate the vascular function in a hypercholesterolemia/atherosclerosis model, in the angiotensin II-dependent 2-kidney 1-clip (2K1C) hypertension model and when both conditions coexist. Eight-week-old apolipoprotein E knockout (apoE; n=20) and C57BL/6 (C57; n=20) mice underwent a 2K1C or sham operation and were studied 28 days later. Mean arterial pressure was higher in apoE-2K1C and C57-2K1C (126+/-3 and 128+/-3 mm Hg) when compared with the apoE-Sham and C57-Sham (103+/-2 and 104+/-2 mm Hg, respectively; P<0.05). The vascular reactivity to norepinephrine (NE; 10(-9) to 2 x 10(-3) mol/L), acetylcholine (ACh), and sodium nitroprusside (SNP; 10(-10) to 10(-3) mol/L) was evaluated in the mesenteric arteriolar bed through concentration-effect curves. NE caused vascular hyper-reactivity in apoE-Sham, apoE-2K1C, and C57-2K1C (maximal response 146+/-5, 144+/-5, and 159+/-4 mm Hg, respectively) compared with C57-Sham (122+/-7 mm Hg; P<0.05). The ACh-induced relaxation was smaller (P<0.05) in apoE-2K1C and C57-2K1C (maximal response 53+/-3% and 46+/-3%) than in apoE-Sham and C57-Sham mice (78+/-5% and 73+/-4%). SNP-induced vascular relaxation showed similar concentration-effect curves in all groups. We conclude that in C57-2K1C mice, the increased reactivity to NE and the decreased endothelium-dependent relaxation contribute to the maintenance of hypertension. The apoE mouse, at early stages of atherosclerosis, shows hyper-reactivity to NE but does not have endothelium dysfunction yet. However, the concurrence of both pathophysiologies does not result in additive effects on the vascular function.     

Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm

We have previously demonstrated that urokinase-type plasminogen activator (uPA) is highly expressed in the aneurysmal segment of the abdominal aorta (AAA) in apolipoprotein E-deficient (apoE-/-) mice treated with angiotensin II (Ang II). In the present study, we tested the hypothesis that uPA is essential for AAA formation in this model. An osmotic minipump containing Ang II (1.44 mg/kg per day) was implanted subcutaneously into 7- to 11-month-old male mice for 1 month. Ang II induced AAA in 9 (90%) of 10 hyperlipidemic mice deficient in apoE (apoE-/-/uPA+/+ mice) but in only 2 (22%) of 9 mice deficient in both apoE and uPA (apoE-/-/uPA-/- mice) (P<0.05). Although the expansion of the suprarenal aorta was significantly less in apoE-/-/uPA-/- mice than in apoE-/-/uPA+/+ mice, the aortic diameters of the aorta immediately above or below the suprarenal aorta were similar between the 2 groups. Ang II induced AAA in 7 (39%) of 18 strain-matched wild-type C57 black/6J control mice. The incidence was significantly higher in atherosclerotic apoE-deficient (apoE-/-) mice, in which 8 (100%) of 8 mice developed AAA. Only 1 (4%) of 27 uPA-/- mice developed AAA after Ang II treatment. We conclude the following: (1) uPA plays an essential role in Ang II-induced AAA in mice with or without preexisting hyperlipidemia and atherosclerosis; (2) uPA deficiency does not affect the diameter of the nonaneurysmal portion of the aorta; and (3) atherosclerosis and/or hyperlipidemia promotes but is not essential for Ang II-induced AAA formation in this model.     

Murine Cytomegalovirus (MCMV) Infection Upregulates P38 MAP kinase in Aortas of Apo E KO Mice: a Molecular Mechanism for MCMV-Induced Acceleration of Atherosclerosis

Multiple studies suggest an association between cytomegalovirus (CMV) infection and atherogenesis; however, the molecular mechanisms by which viral infection might exacerbate atherosclerosis are not well understood. Aortas of MCMV-infected and uninfected Apo E knockout (KO) mice were analyzed for atherosclerotic lesion development and differential gene expression. Lesions in the infected mice were larger and showed more advanced disease compared to the uninfected mice. Sixty percent of the genes in the MAPK pathway were upregulated in the infected mice. p38 and ERK 1/2 MAPK genes were 5.6- and 2.0-fold higher, respectively, in aortas of infected vs. uninfected mice. Levels of VCAM-1, ICAM-1, and MCP-1 were ~2.0–2.6-fold higher in aortas of infected vs. uninfected mice. Inhibition of p38 with SB203580 resulted in lower levels of pro-atherogenic molecules and MCMV viral load in aortas of infected mice. MCMV-induced upregulation of p38 may drive the virus-induced acceleration of atherogenesis observed in our model.     

Extracellular release of the atheroprotective heat shock protein 27 is mediated by estrogen and competitively inhibits acLDL binding to scavenger receptor-A

We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ERbeta)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27o/e) mice were crossed to apoE-/- mice that develop atherosclerosis when fed a high-fat diet. Aortic en face analysis demonstrated a 35% reduction (P < or =0.001) in atherosclerotic lesion area in apoE-/-HSP27o/e mice compared to apoE-/- mice, but primarily in females. Serum -HSP27levels were 10-fold higher in female apoE-/-HSP27o/e mice compared to males, and there was a remarkable inverse correlation between circulating HSP27 levels and lesion area in both male and female mice (r(2)=0.78, P < or =0.001). Mechanistic in vitro studies showed upregulated HSP27 expression and secretion in macrophages treated with estrogen or acLDL. Moreover, exogenous HSP27 added to culture media inhibited macrophage acLDL uptake and competed for the scavenger receptor A (SR-A)--an effect that was abolished with the SR-A competitive ligand fucoidan and absent in macrophages from SR-A-/- mice. Furthermore, extracellular HSP27 decreased acLDL-induced release of the proinflammatory cytokine IL-1beta and increased the release of the antiinflammatory cytokine IL-10. HSP27 is atheroprotective, perhaps because of its ability to compete for the uptake of atherogenic lipids or attenuate inflammation.     

Recombinant CD40L Treatment Protects Allogeneic Murine Bone Marrow Transplant Recipients From Death Caused by Herpes Simplex Virus-1 Infection

Posttransplant infection associated with host immune deficiency isthe major cause of nonrelapse mortality of human bone marrow transplantrecipients. In a new murine model of posttransplant infection,allogeneic bone marrow transplant recipients were infected with herpessimplex virus-1 (HSV-1) via intraperitoneal inoculation 12 weeks aftertransplantation. Allogeneic transplant recipients withgraft-versus-host disease (GVHD) had significantly increased mortalityfrom HSV-1 encephalitis, with deficiencies of both specific anti-HSV-1antibody and total serum IgG2a. GVHD mice displayed a Th2 cytokineprofile (increased interleukin-4 [IL-4] and decreased interferon-)and decreased lipopolysaccharide (LPS) responses, suggesting that bothT-cell and B-cell defects contributed to the impaired production ofantibody. Because passive transfer of hyperimmune serum protected micefrom HSV-1 infection, we hypothesized that CD40 ligand (CD40L), whichinduces B-cell maturation, would protect mice from HSV-1 infection.CD40L-treated GVHD mice showed elevated IgG2a levels and increasedsurvival compared with vehicle-treated transplant recipients.     

Local monocyte chemoattractant protein-1 therapy increases collateral artery formation in apolipoprotein E-deficient mice but induces systemic monocytic CD11b expression,neointimal formation,and plaque progression

Monocyte chemoattractant protein-1 (MCP-1) stimulates the formation of a collateral circulation on arterial occlusion. The present study served to determine whether these proarteriogenic properties of MCP-1 are preserved in hyperlipidemic apolipoprotein E-deficient (apoE-/-) mice and whether it affects the systemic development of atherosclerosis. A total of 78 apoE-/- mice were treated with local infusion of low-dose MCP-1 (1 microg/kg per week), high-dose MCP-1 (10 microg/kg per week), or PBS as a control after unilateral ligation of the femoral artery. Collateral hindlimb flow, measured with fluorescent microspheres, significantly increased on a 1-week high-dose MCP-1 treatment (PBS 22.6+/-7.2%, MCP-1 31.3+/-10.3%; P<0.05). These effects were still present 2 months after the treatment (PBS 44.3+/-4.6%, MCP-1 56.5+/-10.4%; P<0.001). The increase in collateral flow was accompanied by an increase in the number of perivascular monocytes/macrophages on MCP-1 treatment. However, systemic CD11b expression by monocytes also increased, as did monocyte adhesion at the aortic endothelium and neointimal formation (intima/media ratio, 0.097+/-0.011 [PBS] versus 0.257+/-0.022 [MCP-1]; P<0.0001). Moreover, Sudan IV staining revealed an increase in aortic atherosclerotic plaque surface (24.3+/-5.2% [PBS] versus 38.2+/-9.5% [MCP-1]; P<0.01). Finally, a significant decrease in the percentage of smooth muscle cells was found in plaques (15.0+/-5.2% [PBS] versus 5.8+/-2.3% [MCP-1]; P<0.001). In conclusion, local infusion of MCP-1 significantly increases collateral flow on femoral artery ligation in apoE-/- mice up to 2 months after the treatment. However, the local treatment did not preclude systemic effects on atherogenesis, leading to increased atherosclerotic plaque formation and changes in cellular content of plaques.