ABSTRACT
Hypoxia promotes angiogenesis by modulating the transcriptional regulator hypoxia-inducible factor 1alpha (HIF-1alpha). HIF-1alpha is a master regulator of the hypoxic response, and its proangiogenic activities include, but are not limited to, regulation of vascular endothelial growth factor (VEGF). The remodeling of the vasculature during angiogenesis requires an initial destabilization step, which facilitates endothelial sprouting, followed by vessel growth, and restabilization through investment of smooth muscle cells. The complex dynamics of hypoxia-induced angiogenesis prompted us to investigate what aspects of this multi-step process are regulated by HIF-1alpha. To do so, we analyzed the molecular properties of aortic and coronary artery smooth muscle cells in response to forced expression of HIF-1alpha, and by treatment with cobalt chloride, which mimics hypoxia. Our results demonstrate that HIF-1alpha causes a marked reduction in the ability of smooth muscle cells to migrate and adhere to extracellular matrices. Analysis of focal adhesion proteins showed no significant difference in expression or localization of vinculin or focal adhesion kinase (FAK). However, investigation of FAK phosphorylation, a critical mediator of adhesion and migration, revealed tyrosine phosphorylation of FAK is diminished in the presence of HIF-1alpha and cobalt chloride. These results indicate that during hypoxia-induced vessel remodeling, HIF-1alpha functions to dampen adhesion and migration of smooth muscle cells by modulating FAK activity. We suggest that HIF-1alpha expression in smooth muscle cells may augment vessel sprouting by loosening smooth muscle cell attachments to the basement membrane and endothelial cells.
Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Adenoviridae/genetics , Animals , Aorta/cytology , Aorta/metabolism , Aorta/pathology , Blotting, Western , Cattle , Cell Adhesion , Cell Movement , Cells, Cultured , Cobalt/pharmacology , DNA, Complementary/metabolism , Endothelium, Vascular/metabolism , Humans , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunoblotting , Immunoprecipitation , Luciferases/metabolism , Microscopy, Fluorescence , Muscle, Smooth/cytology , Muscle, Smooth/metabolism , Myocytes, Smooth Muscle/cytology , Neovascularization, Pathologic , Phosphorylation , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transfection , Tyrosine/chemistry , Vascular Endothelial Growth Factor A/metabolism , Wound HealingABSTRACT
RATIONALE: Blood platelets have been used extensively as a model system for investigating the role of the serotonin transporter (SERT) in various psychiatric disorders, especially depression. However, to date, it is not known whether platelet serotonin (5-HT) transport would be related to that in brain. OBJECTIVES: We examined 5-HT transport kinetics simultaneously in human blood platelets and human cortical brain synaptosomes to determine whether they were correlated. METHODS: Blood platelets and synaptosomes were obtained from 25 patients undergoing epileptic surgery. Synaptosomes were obtained from normal margins of surgical neuropathology specimens of anterotemporal cortex. RESULTS: Platelet SERT V(max) was significantly correlated with brain SERT V(max) on linear regression (r=0.58, p<0.005), after controlling for the confounding effects of gender (t=-2.4, p=0.025) and time of day (t=2.1, p<0.05). Consistent with previous observations, there was a negative correlation between the maximum velocity (V(max)) of platelet 5-HT transport and pO2 (r=-0.52, p<0.01). Females had a significantly higher pO2 than males (F=4.9, p<0.05). After accounting for gender differences, addition of pO2 did not add further strength to the regression, given the aforementioned gender differences in pO2. The correlation between unadjusted values for platelet vs brain SERT V(max) was r=0.3, p=0.06. CONCLUSIONS: These results suggest that a relationship may exist between 5-HT transport in platelets and cortical synaptosomes, when appropriate controls for confounding factors are employed.
Subject(s)
Blood Platelets/metabolism , Serotonin Plasma Membrane Transport Proteins/metabolism , Serotonin/pharmacokinetics , Synaptosomes/metabolism , Adult , Analysis of Variance , Biological Transport , Brain/metabolism , Female , Humans , Kinetics , Male , Middle Aged , Oxygen/metabolismABSTRACT
The potency of escitalopram ("Lexapro," s-citalopram, LU-26-054) was compared with that of racemic citalopram ("Celexa") using plasma samples from drug-treated normal controls applied to an assay of human serotonin [5-hydroxytryptamine (5-HT)] transport inhibition in blood platelets. Samples were available for both 4-hour and 24-day drug administration. The data indicated that 5-HT transport inhibition was fully manifest for each drug within 4 hours of administration, without significant increase in platelet transport inhibition by 24-day treatment. In addition, a dose-response relationship could be seen for escitalopram and citalopram with increasing 5-HT transport inhibition observed with increasing dose. It was evident from the data that escitalopram was significantly more potent than its racemate in inhibiting human platelet 5-HT transport. Thirty milligrams of escitalopram approximated the effect of 60 mg of racemic citalopram, and similarly, 10 mg of escitalopram approximated that of 20 mg of its racemate. This is the first demonstration of escitalopram's pharmacodynamic effect on the human 5-HT transporter. The results demonstrate its superior potency at the human 5-HT transporter site.
