Vascular endothelium in diabetes

Abstract

Three decades ago a revolutionary idea was born that ascribed to dysfunctional endothelia some manifestations of diabetes, the Steno hypothesis, so named after the Steno Diabetes Center, Gentofte, in Denmark. Here I briefly outline the accomplishments accrued in the past 15 years to buttress this hypothesis. Those include development of novel technological platforms to examine microcirculatory beds, deeper understanding of patterns of microvascular derangement in diabetes, pathophysiology of nitric oxide synthesis and availability, nitrosative and oxidative stress in diabetes, premature senescence of endothelial cells and the role of sirtuin 1 and lysosomal dysfunction in this process, and the state of endothelial glycocalyx and endothelial progenitor cells in diabetes. These pathophysiological findings may yield some therapeutic benefits.

Keywords: endothelial glycocalyx; lysosomal dysfunction; nitric oxide; premature cell senescence; sirtuin 1.

Fig. 1.

The evolution of the Steno hypothesis from its original (A) to our interpretation of the subject in 2001 (B) and my present-day view on endothelial dysfunction in diabetes mellitus (C). A: summary of the original Steno hypothesis that, most importantly, considered albuminuria as a manifestation of widespread microvascular damage. It was hypothesized that this defect is a result of impaired synthesis of what we would today consider endothelial glycocalyx. B: schematic view of hyperglycemia-induced transient and cumulative effects on vascular wall leading to impaired endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) synthesis that in turn affect matrix synthesis and degradation, angiogenesis, and vascular permeability (from Ref. with permission). C: several pathways activated in vascular endothelial cells exposed to hyperglycemia and advanced glycosylation end products (AGEs). Notably, all of these alterations lead to endothelial cell dysfunction manifesting in microalbuminuria. PAI-1, plasminogen activator inhibitor-1; ECM, extracellular matrix; PAR-2, protease-activated receptor-2.

Fig. 2.

Hyperglycemic switch from mitochondrial NO to superoxide production. A: human umbilical vein endothelial cells (HUVEC) respond to the calcium ionophore (A-23187) with an increase in mitochondrial NO production, as detected using diacetate (4-amino-5-methylamino-2′,7′-difluorofuorescein (DAF) fluorescence (after selective loading of DAF in mitochondria). This process is inhibited by elevated ambient d-glucose (but not l-glucose) level. B: pretreatment of HUVEC with a cell-permeable superoxide dismutase (SOD) mimetic, manganese (III) tetrakis (4-benzoic acid)porphyrin chloride (Mn-TBAP), protects mitochondrial NO production against impairment by high d-glucose. C: eNOS expression in mitochondrial fractions (mt) or whole cell lysates (cell) is not affected by high levels of ambient d-glucose. Cytochrome c oxidase (COX) was used as a mitochondrial marker. D: NO production by mitochondria isolated from the livers of Zucker diabetic fat (ZDF) or Zucker lean (ZL) rats. Note that NO generation by mitochondria of ZDF rats is impaired under basal and stimulated conditions (*P < 0.05).

Fig. 3.

Hyperglycemia enhances mitochondrial reactive oxygen species (ROS) production. A: representative images of dichlorodihydrofluorescein diacetate (DDF) colocalizing with MitoTracker-labeled mitochondria in HUVEC. B: HUVEC respond to the calcium ionophore with a minor “hump” in mitochondrial hydrogen peroxide production, as detected using DDF fluorescence (after selective loading of DDF in mitochondria). This process is dramatically enhanced by elevated ambient d-glucose (but not l-glucose) level. C: treatment of HUVEC cultured in 30 mM d-glucose for 24 h with Mn-TBAP abolishes the A-23187-induced increase in ROS production. D: ROS production by mitochondria isolated from livers of ZDF and ZL rats. Note that both the baseline and stimulated ROS production are enhanced in ZDF rats (*P < 0.05).

Fig. 4.

AGE-induced senescence of endothelial cells is receptor for advanced glycation end product (RAGE) dependent. A: representative images of aortic endothelium (top, plain of the aorta; bottom, orifices of intercostal arteries) of ZL and ZDF rats at ages of 8 and 22 wk. B: HUVEC were cultured for 5 days in the presence of native collagen I (NC) or glycosylated long-lived protein collagen I (GC) (100 μM each), and expression of senescence-associated β-galactosidase (SA-β-gal) was quantified. Parallel cultures were exposed to siRAGE or Luciferase GL3 siRNA used as a negative control. siRAGE, but not siGL3, dramatically reduced the proportion of SA-β-gal-positive cells (J. Chen and M. S. Goligorsky, unpublished observations).

Fig. 5.

Expression of sirtuin 1 (SIRT1) in diabetes. A: SIRT1 expression in kidney lysates obtained from db/db and dbm mice at ages 12 (time of a shift from normoglycemia to hyperglycemia) and 30 (time of persistent hyperglycemia) wk. Note that at 30 wk the expression of Sirt1 is reduced. The panel on the bottom summarizes these Western blotting studies. *P < 0.05 (unpublished observations). B: left, results of Western blot analysis of Sirt1 expression after its overexpression (SIRT1), inhibition (shRNA), and control transfection (Mock). Right, results of Sirt1 overexpression or suppression on apoptosis and senescence in cells subjected to GC or NC. *P < 0.05. Reprinted with permission from Ref. .

Fig. 6.

Lysosomal permeabilization by AGEs. A: schematic of the experimental protocol: colocalization of dextran and lysosome-associated protein-1 in control and their dissociation in a permeabilized state. B: glycated collagen induces lysosomal permeabilization in HUVEC. Studies were conducted using dextran-Texas red loading in a lysosomal compartment (followed by immunodetection of LAMP-1 and labeling of nuclei) under control conditions, following application of chloroquine (positive control for permeabilization of the lysosomal membrane) and native and glycated collagen at two different concentrations. A significant and comparable permeabilization of the lysosomal membrane is detected after application of either chloroquine or glycated collagen I.

Fig. 7.

Analysis of bone marrow-derived endothelial progenitors. A-C: fluorescence-activated cell sorting (FACS) analysis (A and C) and direct cell culture of bone marrow-derived cells (B) indicate an increase in the proportion of apoptotic progenitors and a decrease in the number of progenitors detected through lectin⁺/LDL⁺ cells or Sca1⁺/Flk1⁺ cells in db/db mice. Pretreatment of db/db mice in vivo or db/db mice-derived bone marrow cells ex vivo with the organoselenic antioxidant and peroxinitrite scavenger ebselen results in improved viability and restored numbers of bone marrow-derived progenitors. P < 0.05 compared with db/m (*) and with TxCM (#). Reprinted with permission from Ref. .

Fig. 8.

Analysis of acetylcholine-induced vasorelaxation of aortic rings (A) and albuminuria (B) in db/db mice and after treatment with dbm bone marrow-derived cells (Txdb) or db/db bone marrow-derived cells exposed in vivo (Txdb-Ebs in vivo) and ex vivo (Txdb-Ebs ex vivo) to the organoselenic antioxidant and peroxinitrite scavenger ebselen. Note that adoptive transfer of syngeneic bone marrow-derived cells improves macro- and microvasculopathy. P < 0.05 compared with db/db (*), with db/db and Txdb (**), and with TxCM (#). Reprinted with permission from Ref. .