Abstract
The effects of glucose extremes on vascular physiology and endothelial cell function have been examined across a range of time scales. Not unexpectedly, chronic glucose exposure induces long term tissue effects. Yet short term exposure can also impose lasting consequences. The persistence of vascular pathology after euglycemic restoration further suggests a glucose exposure memory. Slow turnover reservoirs such as basement membrane are candidates for prolongation of acute events. We hypothesized that glucose-induced vascular dysfunction is related to altered vasoactive compound handling within the endothelial cell-basement membrane co-regulatory unit. Endothelial cell basement membrane-associated fibroblast growth factor-2 increased linearly with culture glucose within days of elevated glucose exposure. Surprisingly, basement membrane fibroblast growth factor-2 binding kinetics remained unchanged. The glucose-induced increase in basement membrane fibroblast growth factor-2 was instead related to enhanced endothelial cell fibroblast growth factor-2 release and permeability. Cellular fibroblast growth factor-2 release occurred concomitant with apoptosis but was not blocked by caspase inhibitors. These data suggest that release was associated with sub-lethal early apoptotic cell membrane damage, perhaps related to reactive oxygen species formation. High glucose basement membrane in turn enhanced endothelial cell proliferation in a fibroblast growth factor-2-dependent manner. We now show that glucose-induced alterations in endothelial cell function promote changes in basement membrane composition, and these changes further affect endothelial cell function. These data highlight the interrelationship of cell and basement membrane in pathological conditions such as hyperglycemia. These phenomena may explain long term effects on the endothelium of short term exposure to glucose extremes.
Highlights
Unregulated remodeling and abnormal homeostasis of the vasculature
We show that basement membrane fibroblast growth factor-2 (FGF-2) increases linearly with glucose exposure due to endothelial cell function alterations and that the change in basement membrane composition further affects endothelial cell function over an extended time scale
We show that the FGF-2 concentration in the endothelial cell basement membrane increases with both time and glucose exposure
Summary
Cell Culture—Porcine aortic endothelial cells were isolated from Yorkshire swine aortae by the collagenase dispersion method and maintained in low glucose Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 1% penicillin-streptomycin, and 2% glutamine (Invitrogen) [19]. Endothelial cell-released FGF-2 was measured in media collected from cell culture and centrifuged to remove cellular debris. Basement membrane FGF-2 association was measured by adding 5 ng/ml FGF-2 in binding buffer to isolated basement membrane for 0 –360 min This concentration (5 ng/ml) is within the linear binding range and results in physiologically relevant bound FGF-2 levels. FGF-2 was aspirated, basement membrane was washed in binding buffer, and bound FGF-2 was extracted as previously described [22]. Cell permeability to FGF-2 and subsequent binding to basement membrane was measured on cells cultured for 4 days in 5 or 30 mM glucose supplemented media. Samples were centrifuged to pellet cells, washed thoroughly, resuspended in annexin binding buffer, and labeled with annexin V-fluorescein and propidium iodide as per the kit instructions (BD Pharmingen). A value of p Ͻ 0.01 is indicated with an asterisk (*)
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