Reduction of both number and proliferative activity of human endothelial progenitor cells in obesity.

OBJECTIVE: Circulating endothelial progenitor cells (EPCs), responsible for neoangiogenesis and vascular repair, negatively correlate with vascular dysfunction and atherosclerotic risk factors. Because obesity may have a crucial role in the development of endothelial dysfunction, this study evaluated the number and proliferative activity of circulating human EPCs in obese (body mass index (BMI)=48+/-9, n=45) compared with lean (23+/-2, n=45) volunteers. METHODS: EPCs were quantified after isolation of peripheral blood mononuclear cells (PBMCs) using fluorescence-activated cell sorting analyses. In addition, plated PBMCs developed colony-forming units (CFUs) from which 'outgrowth' endothelial cells (OECs) sprouted and differentiated into mature endothelial cells. Growth rates were monitored by periodical microscopic evaluation. Cell-cycle protein expression was determined by western blot analyses. RESULTS: BMI negatively correlated (P<0.01) with the number of CD34(+)/CD133(+)/KDR(+) (r=-0.442), CD34(+)/KDR(+) (r=-0.500) and CD133(+)/KDR(+) (r=-0.282) EPCs. Insulin, leptin, HbA(1c), high-sensitivity C-reactive protein and hypertension, as well as diminished high-density lipoprotein and apolipoprotein A1, were not only associated with obesity but also with significantly reduced EPC levels. Applying selective culture conditions, EPC-CFUs differentiated into OECs that proliferated more slowly when derived from obese compared with lean subjects (obese: 19.9+/-2.2% vs lean: 30.9+/-3.2% grown area per week, P<0.01). The reduced proliferation was reflected by decreased (P<0.05, n=24 for each group) expression of cell-cycle-promoting cyclins and E2F-1, by hypophosphorylation of retinoblastoma protein and by increased (P<0.05, n=24 for each group) expression of the cell-cycle inhibitor p21(WAF-1/Cip1). CONCLUSIONS: Reduced numbers of EPCs along with their premature senescence, as shown in this study, could function as early contributors to the development and progression of vascular dysfunction in obesity.

Insulin does not regulate glucose transport and metabolism in human endothelium.

BACKGROUND: Although endothelial cells express insulin receptors, it is controversially discussed whether the endothelium represents an insulin-responsive tissue. Since available data are primarily restricted to animal endothelial cells, this study tested (i) whether insulin affects glucose metabolism in human endothelium; (ii) whether insulin sensitivity is different in micro- versus macrovascular endothelial cells; and (iii) whether glucose concentration in the incubation medium affects the cells' response to insulin. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs), human adult saphenous vein endothelial cells (HAVECs), human aortic endothelial cells (HAEC), and human retinal endothelial cells (HRECs) as well as human smooth muscle cells were incubated with/without insulin (0.3 nmol L(-1) or 1 micromol L(-1)). Glucose transport, glycogen synthesis, glycogen content, lactate release, and expression of phospho-Akt, Akt, and endothelial nitric oxide synthase (eNOS) were determined. RESULTS: In HUVECs and HRECs, insulin (1 micromol L(-1)) increased (P < 0.05) eNOS expression by ~70% and doubled Akt phosphorylation, but the latter was by far more pronounced in human smooth muscle cells (+1093 +/- 500%, P < 0.05). In human smooth muscle cells, insulin (1 micromol L(-1)) stimulated glycogen synthesis by 67 +/- 11% (P < 0.01). In human micro- (HRECs) and macrovascular endothelial cells (HUVECs, HAVECs and HAECs), insulin, however, failed to stimulate glucose transport, glycogen synthesis, glycogen content, or lactate release under various conditions, i.e. after glucose deprivation or in medium with normal (5.5 mmol L(-1)) or high glucose (30 mmol L(-1)). CONCLUSIONS: Insulin stimulated glycogen synthesis and Akt phosphorylation in human smooth muscle cells. In human micro- and macrovascular endothelial cells, insulin, however, failed to affect glucose uptake and metabolism under all experimental conditions applied, whereas it increased Akt phosphorylation and eNOS expression.

Free fatty acids do not acutely increase asymmetrical dimethylarginine concentrations.

Concentrations of asymmetrical dimethylarginine (ADMA) and free fatty acids (FFAs) are elevated in insulin resistance which is associated with impaired vascular function. We hypothesized that FFAs could alter vascular tone by affecting ADMA concentrations. Plasma FFA levels were increased in seventeen healthy male volunteers by Intralipid/heparin infusion; hemodynamic and biochemical parameters were measured after 90 minutes. Plasma collected before and during Intralipid/heparin or equivalent synthetic FFAs was incubated with human umbilical vein endothelial cells (HUVECs) in vitro. Intralipid/heparin infusion resulted in an approximately seven-fold increase in plasma FFA levels to 1861 +/- 139 micromol/l, which was paralleled by increased systemic blood pressure and forearm blood flow. Intralipid/heparin did not affect ADMA (baseline mean 0.59 [95 % confidence interval [CI]: 0.54; 0.64] and 0.56 [CI: 0.51; 0.59] after 90 minutes), but slightly decreased SDMA (from 0.76, [CI: 0.70; 0.83] to 0.71 [CI: 0.64; 0.74], p < 0.05), and had no effect on ADMA/SDMA ratio. There was no correlation between ADMA and FFA concentrations or forearm blood flow. Incubation of HUVECs with FFA-rich plasma or synthetic FFAs induced an ADMA release after 24 hours, but not after 90 minutes. Acutely increased FFA levels caused hemodynamic effects but did not affect ADMA. Prolonged elevation of FFA levels might influence vascular function by increasing ADMA levels.

Thiazolidinediones inhibit proliferation of microvascular and macrovascular cells by a PPARgamma-independent mechanism.

AIMS/HYPOTHESIS: This study evaluated the hypothesis that peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists, including thiazolidinediones (TZDs) and the rexinoid LG100268 (LG), directly affect human vascular cell function (proliferation, cell cycle, protein expression, lactate release) independently of (1) their PPARgamma-activating potential and (2) the cells' vascular origin. METHODS: Human umbilical vein endothelial cells (HUVECs), human adult vein endothelial cells (HAVECs), human retinal endothelial cells (HRECs) and human retinal pericytes (HRPYCs) were incubated (48 h) with 2-50 micromol/l rosiglitazone (RSG), RWJ241947 (RWJ), pioglitazone (PIO), troglitazone (TRO), 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2) and LG. Proliferation, cell cycle distribution, protein expression, peroxisome proliferator-activated receptor responsive element (PPRE) transcriptional activity and mitochondrial effects were determined by [3H]thymidine incorporation, FACS analyses, western blots, reporter assays and lactate release respectively. RESULTS: In HUVECs, RSG, RWJ, PIO, TRO, PGJ2 and LG reduced (p<0.01) proliferation (due to a G0/G1 cell cycle arrest) by up to 23%, 36%, 38%, 86%, 99% and 93% respectively. The antiproliferative response was similar in HRPYCs and HAVECs, but was attenuated in HRECs. Whereas p21WAF-1/Cip1 and p27Kip were differently affected in HUVECs, all agents reduced (p<0.05) expression of cyclins (D3, A, E, B), cyclin-dependent kinase-2 and hyperphosphorylated retinoblastoma protein. The rank order of the antiproliferative effects of TZDs in HUVECs (RSG approximately PIO approximately RWJ

Expression of uncoupling protein-3 mRNA in rat skeletal muscle is acutely stimulated by thiazolidinediones: an exercise-like effect?

AIMS/HYPOTHESIS: We examined whether thiazolidinediones (TZDs) acutely affect uncoupling protein-3 ( UCP-3) expression in skeletal muscle and plasma NEFA in Sprague-Dawley rats. METHODS: Expression of UCP-3 mRNA in hindlimb muscles and plasma NEFA were measured after a single intraperitoneal injection of TZDs in healthy male rats. RESULTS: Independent of which TZD was injected (50 micromol/kg), UCP-3 expression in gastrocnemius muscle was distinctly increased after 6 h (increase vs vehicle-injected control: pioglitazone, 10.3+/-3.2-fold, p=0.03; rosiglitazone, 8.7+/-1.2-fold, p=0.001; RWJ241947, 9.5+/-2.7-fold, p=0.03). This was accompanied by elevated plasma NEFA (control 158+/-13 micromol/l; pioglitazone, 281+/-40 micromol/l, p=0.03; rosiglitazone, 276+/-27 micromol/l, p=0.005; RWJ241947, 398+/-51 micromol/l, p=0.004). The increase in plasma NEFA could in part have mediated TZD-induced UCP-3 expression, but increased UCP-3 mRNA was also found in isolated muscle after 2 h of TZD exposure in vitro (25 micromol/l pioglitazone, 1.7+/-0.3-fold, p=0.046), suggesting that TZDs act directly and independently of NEFA on skeletal muscle. CONCLUSIONS/INTERPRETATION: In healthy rats, a single dose of TZDs rapidly increases UCP-3 mRNA in skeletal muscle and plasma NEFA. This effect resembles the acute response to a bout of exercise.

Modulation by leptin of proliferation and apoptosis in vascular endothelial cells.

AIM: Plasma leptin concentrations not only correlate with body fat mass, but also with the degree of hypertensive retinopathy. The present study was designed to further examine, whether leptin's proliferative, proangiogenic activity relates to a yet uncovered anti-apoptotic effect. RESULTS: Leptin (10-50 nmol/l) concentration-dependently reduced apoptosis in HUVECs (human umbilical vein endothelial cells), HAVECs (human adult vein endothelial cells) and HMECs (human microvascular endothelial cells) by 20% (P < or = 0.05). These findings were supported by increased expression of the apoptosis inhibitor bcl-2 (+55%, P < or = 0.05) as well as by differential modulation of the respective cell cycle checkpoint genes/proteins p53 (-20%, P < or = 0.01), p21(WAF-1/Cip1) (-23%, P < or = 0.05) and the Retinoblastoma protein (+123%, P < or = 0.01). CONCLUSION: bcl-2 dependent anti-apoptotic action might contribute to leptin's proangiogenic activity and thereby promote the development of vascular proliferative disease in obesity.

Levamisole induced apoptosis in cultured vascular endothelial cells.

To better understand the anticancer activity of Levamisole (LMS), which serves as an adjuvant in colon cancer therapy in combination with 5-Fluorouracil, this study analyses LMS' ability to induce apoptosis and growth arrest in cultured human micro- and macrovascular endothelial cells (ECs) and fibroblasts. Cells exposed (24 h) to Levamisole (range: 0.5 - 2 mmol l(-1)) alone or in combination with antioxidants (10 mmol l(-1) glutathione or 5 mmol l(-1) N-Acetylcysteine or 0.1 mmol l(-1) Tocopherol) were evaluated for apoptosis ((3)H-thymidine assays, in situ staining), mRNA/protein expression (Northern/Western blot), and proliferation ((3)H-thymidine incorporation). Levamisole dose-dependently increased apoptosis in ECs to 230% (HUVECs-human umbilical vein ECs), 525% (adult human venous ECs) and 600% (human uterine microvascular ECs) but not in fibroblasts compared to control cells (set as 100%). Levamisole increased in ECs integrin-dependent matrix adhesion, inhibited proliferation (-70%), reduced expression of survival factors such as clusterin (-30%), endothelin-1 (-43%), bcl-2 (-34%), endothelial NO-synthase (-32%) and pRb (Retinoblastoma protein: -89%), and increased that of growth arrest/death signals such as p21 (+73%) and bak (+50%). LMS (2 mmol l(-1))-induced apoptosis was inhibited by glutathione (-50%) and N-Acetylcysteine (-36%), which also counteracted reduction by Levamisole of pRb expression, suggesting reactive oxygen species and pRb play a role in these processes. The ability of LMS to selectively induce apoptosis and growth arrest in endothelial cells potentially hints at vascular targeting to contribute to Levamisole's anticancer activity.