Cytomegalovirus infection leads to microvascular dysfunction and exacerbates hypercholesterolemia-induced responses

Abstract

Cytomegalovirus (CMV) persistently infects more than 60% of the worldwide population. In immunocompetent hosts, it has been implicated in several diseases, including cardiovascular disease, possibly through the induction of inflammatory pathways. Cardiovascular risk factors promote an inflammatory phenotype in the microvasculature long before clinical disease is evident. This study determined whether CMV also impairs microvascular homeostasis and synergizes with hypercholesterolemia to exaggerate these responses. Intravital microscopy was used to assess endothelium-dependent and -independent arteriolar vasodilation and venular leukocyte and platelet adhesion in mice after injection with either mock inoculum or murine CMV (mCMV). Mice were fed a normal (ND) or high-cholesterol (HC) diet beginning at 5 weeks postinfection (p.i.), or a HC diet for the final 4 weeks of infection. mCMV-ND mice exhibited impaired endothelium-dependent vasodilation versus mock-ND at 9 and 12 weeks and endothelium-independent arteriolar dysfunction by 24 weeks. Transient mild leukocyte adhesion occurred in mCMV-ND venules at 7 and 21 weeks p.i. HC alone caused temporary arteriolar dysfunction and venular leukocyte and platelet recruitment, which were exaggerated and prolonged by mCMV infection. The time of introduction of HC after mCMV infection determined whether mCMV+HC led to worse venular inflammation than either factor alone. These findings reveal a proinflammatory influence of persistent mCMV on the microvasculature, and suggest that mCMV infection enhances microvasculature susceptibility to both inflammatory and thrombogenic responses caused by hypercholesterolemia.

Figure 1

Arteriolar vasodilation responses to acetylcholine (ACh) (A) or papaverine (papav) (B) in mock-inoculated or mCMV-infected mice maintained on a normal diet (ND). Measurements (expressed as % change in diameter from baseline) were performed at different time points between 4 and 24 weeks p.i. The data in this figure are the combined ND data from both substudies. *P < 0.01 versus corresponding Mock-ND; **P < 0.005 versus corresponding Mock-ND.

Figure 2

Leukocyte adhesion in postcapillary venules (#/mm² vessel surface) (A) and emigration into the interstitium (#/mm² interstitium) (B) measured in mock-inoculated or mCMV-infected mice maintained on a normal diet (ND) between 4 and 24 weeks p.i. The data shown are the combined data from all ND mice at all time points from the two substudies, which are represented separately in Figures 5 and 7. *P < 0.05 versus corresponding Mock-ND; **P < 0.01 versus corresponding Mock-ND.

Figure 3

Time course of platelet recruitment in postcapillary venules of mice injected with mock inoculum or mCMV at wk 0. Platelet interactions are expressed as saltation (transient; A), firm adhesion (≥30 s; B), or total adhesion (C; #/mm² vessel surface). The ND data collected at all time points in the two substudies are shown jointly here

Figure 4

Endothelium-dependent (A) and -independent (B) vasodilatory responses to ACh or Papav, respectively in arterioles of mice exposed to mock inoculum or mCMV and maintained on a normal diet (ND) or placed on a high-cholesterol diet (HC) at 5 weeks p.i. before observation at 7, 9, 13, 17, or 21 weeks p.i. Data here are also represented in Figure 1. *P < 0.05 mCMV-ND and mCMV-HC versus Mock-ND; ^†P < 0.01 mCMV-ND and mCMV-HC versus Mock-HC; ^‡P < 0.05 mCMV-HC versus Mock-ND; ^§P < 0.05 versus all other groups; ^¶P < 0.05 mCMV-HC versus mCMV-ND (comparisons made within each time point).

Figure 5

Leukocyte adhesion (#/mm² vessel surface) (A) and emigration (#/mm² interstitium) (B), and total platelet adhesion (#/mm² vessel surface) (C) in postcapillary venules of mice injected with mock inoculum or mCMV at Day 0. Mice were either kept on normal chow (ND) for the entire period or changed to a high-cholesterol diet (HC) at 5 weeks p.i. before observation at 7, 9, 13, 17, or 21 weeks p.i. Data here are also represented in Figures 2 and 3. *P < 0.05 Mock-ND versus all other groups; ^†P < 0.05 mCMV-HC versus mCMV-ND; ^‡P < 0.05 versus all other groups; ^§P < 0.001 versus all other groups; ^¶P < 0.05 mCMV-HC versus Mock-ND; ^∥P < 0.01 Mock-HC versus Mock-ND (comparisons made within each time point).

Figure 6

Vasodilation responses to ACh (endothelium-dependent) (A) or papav (endothelium-independent) (B) measured in arterioles of mice over a 6-month period after injection with mock inoculum or mCMV. Mice from both groups were maintained on a normal diet (ND) for the entire period or switched to a high-cholesterol diet (HC) for the final 4 weeks before observation at 4, 6, 9, 12, 18, and 24 weeks p.i. Data here are also represented in Figure 1. *P < 0.05 mCMV-ND and mCMV-HC versus Mock-ND; ^†P < 0.05 mCMV-HC versus Mock-HC; ^‡P < 0.01 mCMV-ND and mCMV-HC versus Mock-HC; ^§P < 0.05 mCMV-HC versus Mock-ND; ^¶P < 0.05 mCMV-ND versus both Mock groups; ^∥P < 0.05 mCMV-ND versus Mock-ND (comparisons made within each time point).

Figure 7

Blood cell recruitment in postcapillary venules of mice injected with mock inoculum or infected with mCMV and kept on a normal diet (ND) or placed on a high-cholesterol diet (HC) for the final 4 weeks before observation. Leukocyte adhesion (#/mm² vessel surface) (A) and emigration (#/mm² interstitium) (B), and platelet adhesion (#/mm² vessel surface) (C) were measured at 4, 6, 9, 12, 18, and 24 weeks after infection. Data here are also represented in Figures 2 and 3. *P < 0.05 mCMV-HC versus all other groups; ^†P < 0.005 mCMV-HC versus all other groups; ^‡P ≤ 0.0001 mCMV-HC versus all other groups; ^§P < 0.05 mCMV-HC versus Mock-ND and mCMV-ND; ^¶P < 0.05 mCMV-HC versus Mock-HC and mCMV-ND; ^∥P < 0.05 mCMV-HC versus Mock-ND (comparisons made within each time point).