Azithromycin reduces Chlamydia pneumoniae-induced attenuation of eNOS and cGMP production by endothelial cells.
BACKGROUND
Intracellular infections with cytomegalovirus (CMV) or Chlamydia pneumoniae (Cp) may play a role in the aetiology of atherosclerosis. Nitric oxide (NO) is a key regulator of endothelial function. Under pathological conditions uncoupling of endothelial nitric oxide synthase (eNOS) leads to vessel damage as a result of production of oxygen radicals instead of NO. We hypothesized that infection-induced atherosclerosis is initiated by changes in NO metabolism and may be reversed by azithromycin treatment.


METHODS
Confluent human umbilical vein endothelial cells (HUVECs) were infected with Cp or CMV. After 48 h of infection, production of eNOS, cyclic guanosine monophosphate (cGMP) and reactive oxygen species (ROS) was measured. Detection of cGMP was used as a reporter assay for the bioavailability of NO. Subsequently, Cp- and CMV-infected HUVECs were coincubated with 0.016 mg L(-1) and 1 mg L(-1) azithromycin.


RESULTS
Infection with Cp (MOI 1 and MOI 0.1) and CMV (MOI 1) caused a dose- and time-dependent reduction of eNOS production in the HUVECs: Cp MOI 1: 1141 +/- 74 pg mL(-1) (P < 0.01); Cp MOI 0.1: 3189 +/- 30 pg mL(-1) (P < 0.01); CMV: 3213 +/- 11 pg mL(-1) (P < 0.01) vs. 3868 +/- 83 pg mL(-1) for uninfected HUVECs. Chlamydia pneumoniae- but not CMV-infection also reduced cGMP-production (Cp: 0.195 +/- 0.030 pmol mL(-1) (P < 0.01); CMV: 0.371 +/- 27 pmol mL(-1) (P > 0.05) vs. 0.378 +/- 0.019 pmol mL(-1) for uninfected HUVECs). CMV-infection did not affect ROS production either, but Cp-infection reduced ROS-production by 21% (P > 0.05; Cp MOI 0.1) to 68% (P < 0.01; Cp MOI 1). Azithromycin treatment restored Cp-induced eNOS, cGMP and ROS production in a dose-dependent manner.


CONCLUSIONS
Infection with Cp in endothelial cells in vitro attenuates eNOS, cGMP and ROS production in HUVECs and azithromycin reverses Cp-induced effects on eNOS, cGMP and ROS-production. The results from our in vitro research support the role of antibiotic therapy for infection-induced atherosclerosis by indicating that azithromycin does actually improve endothelial function.
Bouwman
J J M
JJ
Department of Medical Microbiology and Immunology, Diakonessen Hospital Utrecht, Utrecht, The Netherlands. jbouwman@diakhuis.nl
Visseren
F L J
FL
Bevers
L M
LM
van der Vlist
W E
WE
Bouter
K P
KP
Diepersloot
R J A
RJ
D000900:Anti-Bacterial Agents / Q000627:therapeutic use*; D017963:Azithromycin / Q000627:therapeutic use*; D002470:Cell Survival; D002478:Cells, Cultured; D023521:Chlamydophila Infections / Q000188:drug therapy* / Q000378:metabolism; D016993:Chlamydophila pneumoniae; D006152:Cyclic GMP / Q000096:biosynthesis*; D003586:Cytomegalovirus Infections / Q000188:drug therapy / Q000378:metabolism; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis*; D017382:Reactive Oxygen Species / Q000378:metabolism
D000900:Anti-Bacterial Agents; D017382:Reactive Oxygen Species; D017963:Azithromycin; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP