Diabetes impairs the vascular recruitment of normal stem cells by oxidant damage, reversed by increases in pAMPK, heme oxygenase-1, and adiponectin - PubMed (original) (raw)

. 2009 Feb;27(2):399-407.

doi: 10.1634/stemcells.2008-0800.

Silvia Morbelli, Luca Vanella, Claudia Kusmic, Cecilia Marini, Michela Massollo, Carla Augeri, Mirko Corselli, Chiara Ghersi, Barbara Chiavarina, Luigi F Rodella, Antonio L'Abbate, George Drummond, Nader G Abraham, Francesco Frassoni

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Free PMC article

Diabetes impairs the vascular recruitment of normal stem cells by oxidant damage, reversed by increases in pAMPK, heme oxygenase-1, and adiponectin

Gianmario Sambuceti et al. Stem Cells. 2009 Feb.

Free PMC article

Abstract

Background: Atherosclerosis progression is accelerated in diabetes mellitus (DM) by either direct endothelial damage or reduced availability and function of endothelial progenitor cells (EPCs). Both alterations are related to increased oxidant damage.

Aim: We examined if DM specifically impairs vascular signaling, thereby reducing the recruitment of normal EPCs, and if increases in antioxidant levels by induction of heme oxygenase-1 (HO-1) can reverse this condition.

Methods: Control and diabetic rats were treated with the HO-1 inducer cobalt protoporphyrin (CoPP) once a week for 3 weeks. Eight weeks after the development of diabetes, EPCs harvested from the aorta of syngenic inbred normal rats and labeled with technetium-99m-exametazime were infused via the femoral vein to estimate their blood clearance and aortic recruitment. Circulating endothelial cells (CECs) and the aortic expression of thrombomodulin (TM), CD31, and endothelial nitric oxide synthase (eNOS) were used to measure endothelial damage.

Results: DM reduced blood clearance and aortic recruitment of EPCs. Both parameters were returned to control levels by CoPP treatment without affecting EPC kinetics in normal animals. These abnormalities of EPCs in DM were paralleled by reduced serum adiponectin levels, increased numbers of CECs, reduced endothelial expression of phosphorylated eNOS, and reduced levels of TM, CD31, and phosphorylated AMP-activated protein kinase (pAMPK). CoPP treatment restored all of these parameters to normal levels.

Conclusion: Type II DM and its related oxidant damage hamper the interaction between the vascular wall and normal EPCs by mechanisms that are, at least partially, reversed by the induction of HO-1 gene expression, adiponectin, and pAMPK levels.

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Figures

Figure 1

Figure 1

EPC kinetics in blood. (A): Time concentration curves of radioactivity in the blood are shown expressed as a percentage of the dose (and thus, of the labeled injected EPCs). Diabetes was associated with a prolonged persistence of EPCs in the blood, as seen by the higher values of radioactivity (and thus, of EPC concentration) at each time point. Induction of heme oxygenase-1 (HO-1) gene expression by CoPP treatment (gray symbols) selectively accelerated EPC clearance from the blood in diabetic animals (squares), without an effect on nondiabetic rats (circles). (B): Whole body clearance of EPCs. Diabetes was associated with a marked reduction in EPC recruitment in the body, expressed by a prolonged persistence of these cells in the blood (**p < .01), whereas CoPP treatment restored this variable to normal values. Abbreviations: CoPP, cobalt protoporphyrin; EPC, endothelial progenitor cell.

Figure 2

Figure 2

EPC recruitment rate in the aorta, lung, spleen, and liver. The recruitment rate was unaffected by diabetes and CoPP treatment in the liver and spleen. Reciprocal results are seen in the aorta and lung of diabetic animals. **p < .001, diabetic rats versus controls. Abbreviations: CoPP, cobalt protoporphyrin; EPC, endothelial progenitor cell.

Figure 3

Figure 3

Molecular effects of CoPP treatment. (A): Enzyme-linked immunosorbent assay for the presence of serum oxidized proteins. CoPP restored normal values of oxidized proteins in the serum of diabetic rats; n = 5, *p < .01 versus control, **p < .01 versus diabetes. (B): Heme-oxygenase activity in the aorta, n = 4. CoPP treatment markedly increased Hemeoxygenase activity in diabetic rats, **p < .001 versus control and diabetic animals. (C): Adiponectin levels in the plasma of control rats, diabetic rats, and diabetic rats treated with CoPP. CoPP markedly increased serum concentration of this hormone in diabetes: n = 4. *p < .05 control versus Diabetic and control rats.

Figure 4

Figure 4

Immunohistochemical staining of TM (A) and CD31 (B) from control and diabetic animals. (C): Optical density analysis of TM staining in diabetic rats and its restoration by CoPP. *p < .05, control versus diabetic animals, mean ± standard error, n = 40. Abbreviations: CoPP, cobalt protoporphyrin; CTR, control; I, tunica intima; IOD, integrated optical density; M, tunica media; TM, thrombomodulin.

Figure 5

Figure 5

Reduction of endothelial damage by CoPP treatment. (A): The number of CECs increased significantly in STZ-induced diabetic rats (p < .05) relative to control rats. Administration of the heme oxygenase-1 inducer CoPP decreased the number of CECs, n = 6. *p < .05 versus controls; #p < .05 versus diabetic rats. (B): Similarly, endothelial cell membrane fragments in blood obtained from control rats were increased in diabetic rats and decreased after CoPP treatment, n = 5. *p < .001 versus controls; #p < .001 versus diabetic rats. Abbreviations: CEC, circulating endothelial cell; CoPP, cobalt protoporphyrin; STZ, streptozotocin.

Figure 6

Figure 6

Effect of CoPP treatment on endothelial gene expression. (A): Western blot and densitometry analysis of eNOS and peNOS from control, diabetic, and CoPP-treated diabetic rat aortas. Quantitative densitometry evaluation of eNOS and peNOS in the aorta was determined. Each bar represents the mean ± standard error of four experiments. *p < .001 for diabetic versus CoPP-treated diabetic rats. (B): Effect of diabetes and HO-1 expression on pAKT and total AKT in the aorta's proteins, and AMPK and pAMPK. Quantitative densitometry evaluations in aorta tissue homogenates of pAKT and pAMPK are expressed as a ratio to HO-2. *p < .01, diabetic rats versus control rats or CoPP-treated diabetic rats. Abbreviations: AKT, phosphatidylinositol 3-kinase; AMPK, AMP-activated protein kinase; CoPP, cobalt protoporphyrin; eNOS, endothelial nitric oxide synthase; HO, heme oxygenase; p, phosphorylated.

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