Functional Activities Of Endothelial Progenitor Cells Research Articles

Direct renin inhibition with aliskiren improves ischemia-induced neovascularization: Blood pressure-independent effect

BackgroundRenin is the rate limiting step for the activation of the renin-angiotensin-aldosterone system, which is linked to the development of endothelial dysfunction, hypertension and atherosclerosis. However, the specific role of renin during physiological responses to tissue ischemia is currently unknown. Aliskiren is the only direct renin inhibitor that is clinically used as an orally active antihypertensive drug. Here we tested the hypothesis that aliskiren might improve neovascularization in response to ischemia. Methods and resultsAt a dose that did not modulate blood pressure (10 mg/kg), aliskiren led to improved blood flow recovery after hindlimb ischemia in C57BL/6 mice (Doppler flow ratios 0.71 ± 0.07 vs. 0.55 ± 0.03; P < 0.05). In ischemic muscles, treatment with aliskiren was associated with a significant increase of vascular density, reduced oxidative stress levels and increased expression of VEGF and eNOS. Aliskiren treatment also significantly increased the number of bone marrow-derived endothelial progenitor cells (EPCs) after hindlimb ischemia. Moreover, the angiogenic properties of EPCs (migration, adhesion, integration into tubules) were significantly improved in mice treated with aliskiren. In vitro, aliskiren improves cellular migration and tubule formation in HUVECs. This is associated with an increased expression of nitric oxide (NO), and a significant reduction of oxidative stress levels. Importantly, the angiogenic properties of aliskiren in vitro and in vivo are completely abolished following treatment with the NOS inhibitor l-NAME. ConclusionDirect renin inhibition with aliskiren leads to improved ischemia-induced neovascularization that is not dependant on blood pressure lowering. The mechanism involves beneficial effects of aliskiren on oxidative stress and NO angiogenic pathway, together with an increase in the number and the functional activities of EPCs.

Abstract 15630: Direct Renin Inhibition With Aliskiren Improves Ischemia-Induced Neovascularization

Background: The activation of the renin-angiotensin system is associated with impaired formation of new blood vessels (neovascularization) in response to ischemia. Aliskiren is the only direct renin inhibitor that is clinically used as an orally active antihypertensive drug. Here we tested the hypothesis that aliskiren might improve neovascularization following ischemia. Methods and Results: C57BL/6 mice were treated with a high dose of aliskiren (50 mg/kg), a low dose of aliskiren (10 mg/kg), or drinking water only. After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. Treatment with aliskiren led to a significantly faster rate of blood flow recovery after hindlimb ischemia (Laser Doppler). Interestingly the lower dose of aliskiren, which did not reduce blood pressure, provided similar improvement of blood flow recuperation compared to the higher dose of aliskiren. At day 21 after surgery, Doppler flow ratios were significantly improved in mice treated with aliskiren (0.69+/-0.07 vs. 0.52+/-0.03; p&lt;0.05). This was associated with an increased expression of angiogenic factors in ischemic muscles, including VEGF and eNOS. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascularisation. We found that aliskiren significantly increased the number of bone marrow EPCs at day 7 after ischemia (172+/-7% increase; p&lt;0.05). Moreover, the adhesive properties of EPCs were significantly improved in mice treated with aliskiren (175+/-5% increase; p&lt;0.05). In vitro, aliskiren improves cellular migration and tubule formation in HUVECs. This is associated with an increased expression of nitric oxide (DAF staining), and a significant reduction of oxidative stress levels (DHE staining). Importantly, the antioxidant and angiogenic properties of aliskiren in HUVECs are abolished following treatment with the NOS inhibitor L-NAME. Conclusions: Direct renin inhibition with aliskiren leads to improved ischemia-induced neovascularization that is not dependant on blood pressure lowering. The mechanisms involve beneficial effects of aliskiren on NO and angiogenic pathways in ischemic tissues, together with an increase in the number and the functional activity of EPCs.

GroEL1, a Heat Shock protein 60 of Chlamydia pneumoniae, Impairs Neovascularization by Decreasing Endothelial Progenitor Cell Function

The number and function of endothelial progenitor cells (EPCs) are sensitive to hyperglycemia, hypertension, and smoking in humans, which are also associated with the development of atherosclerosis. GroEL1 from Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis. However, the actual effects of GroEL1 on EPC function are unclear. In this study, we investigate the EPC function in GroEL1-administered hind limb-ischemic C57BL/B6 and C57BL/10ScNJ (a toll-like receptor 4 (TLR4) mutation) mice and human EPCs. In mice, laser Doppler imaging, flow cytometry, and immunohistochemistry were used to evaluate the degree of neo-vasculogenesis, circulating level of EPCs, and expression of CD34, vWF, and endothelial nitric oxide synthase (eNOS) in vessels. Blood flow in the ischemic limb was significantly impaired in C57BL/B6 but not C57BL/10ScNJ mice treated with GroEL1. Circulating EPCs were also decreased after GroEL1 administration in C57BL/B6 mice. Additionally, GroEL1 inhibited the expression of CD34 and eNOS in C57BL/B6 ischemic muscle. In vitro, GroEL1 impaired the capacity of differentiation, mobilization, tube formation, and migration of EPCs. GroEL1 increased senescence, which was mediated by caspases, p38 MAPK, and ERK1/2 signaling in EPCs. Furthermore, GroEL1 decreased integrin and E-selectin expression and induced inflammatory responses in EPCs. In conclusion, these findings suggest that TLR4 and impaired NO-related mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of GroEL1 from C. pneumoniae.

Fish oil-enriched diet protects against ischemia by improving angiogenesis, endothelial progenitor cell function and postnatal neovascularization

BackgroundFish oil consumption has been associated with a reduced incidence of cardiovascular diseases. However, the precise mechanisms involved are not completely understood. Here we tested the hypothesis that a fish oil-enriched diet improves neovascularization in response to ischemia. Methods and resultsC57Bl/6 mice were fed a diet containing either 20% fish oil, rich in long-chain n-3 polyunsaturated fatty acids (PUFAs), or 20% corn oil, rich in n-6 PUFAs. After 4 weeks, hindlimb ischemia was surgically induced by femoral artery removal. We found that blood flow recovery was significantly improved in mice fed a fish oil diet compared to those fed a corn oil diet (Doppler flow ratio (DFR) at day 21 after surgery 78 ± 5 vs. 56 ± 4; p < 0.01). Clinically, this was associated with a significant reduction of ambulatory impairment and ischemic damage in the fish oil group. At the microvascular level, capillary density was significantly improved in ischemic muscles of mice fed a fish oil diet. This correlated with increased expression of VEGF and eNOS in ischemic muscles, and higher NO concentration in the plasma. Endothelial progenitor cells (EPCs) have been shown to have an important role for postnatal neovascularization. We found that the number of EPCs was significantly increased in mice fed a fish oil diet. In addition, oxidative stress levels (DCF-DA, DHE) were reduced in EPCs isolated from mice exposed to fish oil, and this was associated with improved EPC functional activities (migration and integration into tubules). In vitro, treatment of EPCs with fish oil resulted in a significant increase of cellular migration. In addition, the secretion of angiogenic growth factors including IL6 and leptin was significantly increased in EPCs exposed to fish oil. ConclusionFish oil-enriched diet is associated with improved neovascularization in response to ischemia. Potential mechanisms involved include activation of VEGF/NO pathway in ischemic tissues together with an increase in the number and the functional activities of EPCs.

034 Endothelial Nitric Oxide Synthase-Overexpressing Human Early Outgrowth Endothelial Progenitor Cells Decrease Human Coronary Artery Smooth Muscle Cell Migration Through Paracrine Functions

Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that overexpressing endothelial nitric oxide synthase (eNOS), or treatment with estrogen, would potentiate the beneficial effects of EPCs in the context of restenosis. We found that native human early outgrowth EPCs (hEPCs) did not have any effect on human coronary artery smooth muscle cell (HCASMC) proliferation and migration In vitro, evaluated by BrdU incorporation and wound scratch assay respectively. In contrast, the NO donor SNAP significantly decreased the proliferation and migration of HCASMCs. Thereafter, hEPCs were either transfected with a human eNOS plasmid or stimulated with 17β-estradiol (E2) prior to being co-cultured with HCASMCs. Total eNOS protein and eNOS phosphorylation levels were increased by 3- to 3.5-fold in eNOS-transfected or E2-stimulated hEPCs, evaluated by western blot. This was associated with a 3-fold increase in NO production, performed by DAF-FM diacetate immunofluorescence (p<0.05). In eNOS-overexpressing hEPCs, enhanced bcl-2/bax ratio and reduced Annexin V/propidium iodide labeling indicated increased survival. Interestingly, we observed a significant (p<0.05) decrease in HCASMC migration when co-cultured with eNOS-overexpressing hEPCs, by 23%, or with E2-stimulated hEPCs, by 56%. However, HCASMC proliferation was not affected by either eNOS-overexpressing or E2-stimulated hEPCs.These results suggest that overexpressing eNOS in hEPCs increases their survival and enhances their capacity to modulate HCASMC migration through paracrine effects.Réseau de thérapie cellulaire et tissulaire Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that overexpressing endothelial nitric oxide synthase (eNOS), or treatment with estrogen, would potentiate the beneficial effects of EPCs in the context of restenosis. We found that native human early outgrowth EPCs (hEPCs) did not have any effect on human coronary artery smooth muscle cell (HCASMC) proliferation and migration In vitro, evaluated by BrdU incorporation and wound scratch assay respectively. In contrast, the NO donor SNAP significantly decreased the proliferation and migration of HCASMCs. Thereafter, hEPCs were either transfected with a human eNOS plasmid or stimulated with 17β-estradiol (E2) prior to being co-cultured with HCASMCs. Total eNOS protein and eNOS phosphorylation levels were increased by 3- to 3.5-fold in eNOS-transfected or E2-stimulated hEPCs, evaluated by western blot. This was associated with a 3-fold increase in NO production, performed by DAF-FM diacetate immunofluorescence (p<0.05). In eNOS-overexpressing hEPCs, enhanced bcl-2/bax ratio and reduced Annexin V/propidium iodide labeling indicated increased survival. Interestingly, we observed a significant (p<0.05) decrease in HCASMC migration when co-cultured with eNOS-overexpressing hEPCs, by 23%, or with E2-stimulated hEPCs, by 56%. However, HCASMC proliferation was not affected by either eNOS-overexpressing or E2-stimulated hEPCs. These results suggest that overexpressing eNOS in hEPCs increases their survival and enhances their capacity to modulate HCASMC migration through paracrine effects. Réseau de thérapie cellulaire et tissulaire

Protection against vascular aging in Nox2-deficient mice: Impact on endothelial progenitor cells and reparative neovascularization

BackgroundAging is associated with increased oxidative stress levels and impaired neovascularization following ischemia. Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in the context of aging. Methods and resultsHindlimb ischemia was surgically induced by femoral artery removal in young (2 months) and old (10 months) Nox2-deficient (Nox2−/−) and wild type mice. We found that Nox2 expression is increased by aging in ischemic muscles of wild type mice. This is associated with a significant reduction of blood flow recovery after ischemia in old compared to young mice at day 21 after surgery (Doppler flow ratios: 0.51 ± 0.05 vs. 0.72 ± 0.05; p < 0.05). We also demonstrate that capillary and arteriolar densities are significantly reduced in ischemic muscles of old animals, while oxidative stress levels are increased (nitrotyrosine immunostaining). Importantly, Nox2 deficiency reduces oxidative stress levels in ischemic tissues and restores blood flow recuperation and vascular densities in old animals. Endothelial progenitor cells (EPCs) have an important role for postnatal neovascularization. Here we show that the functional activities of EPCs (migration, adhesion to mature endothelial cells) are significantly impaired in old compared to young mice. However, Nox2 deficiency rescues EPC functional activities in old animals. We also demonstrate an age-dependent pathological increase of oxidative stress levels in EPCs (DHE, DCF-DA) that is not present in Nox2-deficient animals. ConclusionNox2-containing NADPH oxidase deficiency protects against age-dependent impairment of neovascularization. Potential mechanisms include reduced ROS generation in ischemic tissues and preserved angiogenic activities of EPCs.

Effect of lisinopril on circulating endothelial progenitor cells in patients with essential hypertention

Objective. To assess the effect of lisinopril on the dynamics of circulating endothelial progenitor cells (EPC) as well as the clinical markers of cardiovascular risk in patients with essential hypertension. Design and methods. We have studied the number and functional activity of EPC and clinical markers of cardiovascular risk in carefully selected group of 18 patients with essential hypertension, who have never been treated by angiotensin converting enzyme inhibitors (ACEi), and 19 normotensive control subjects. Hypertensive patients took lisinopril in dose 10-20 mg daily and then were re-examined after 12 weeks of lisinopril therapy. Results. The present study shows that hypertensive patients are characterized by a relative decline in the number of circulating EPC in conditions of endothelial dysfunction. Also the cells with angiogenic potential have a decreased proliferative capacity in hypertensive patients in comparison with healthy controls (1,4 ± 0,9 vs 2,2 ± 1,2 units/mm 2 ; p < 0,01). Changes of the functional activity of EPC are closely associated with pathological vascular remodeling. Conclusions. ACEi lisinopril improves functional properties of the endothelium in hypertensive patients through mobilizing effect on the EPC and stimulates their proliferation activity.

Ginkgolide B promotes proliferation and functional activities of bone marrow-derived endothelial progenitor cells: involvement of Akt/eNOS and MAPK/p38 signaling pathways

Bone marrow-derived, circulating endothelial progenitor cells (EPCs) contribute to neovascularization in various diseases, and represent a very interesting alternative cell source for enhancing vasculogenesis in regenerative medicine. In this study, we investigated the effects of Ginkgolide B (GB) on proliferation and differentiation of EPCs, and the involved signaling pathway in vitro. EPC proliferation, migration, adhesion and angiogenesis activities were assessed with the WST-8 assay, Transwell chamber assay, cell counting and angiogenesis kit, respectively. Apoptosis was detected with annexin V and propidium iodide staining. The protein expression of angiogenesis-related makers was detected by Western blot, and related gene expression was determined by real-time polymerase chain reaction (RT-PCR). The results showed that GB promoted the proliferation and endothelial gene expression, and markedly enhanced vascular endothelial growth factor-induced migration response and the capability to incorporate into the vascular networks in EPCs. GB protected EPCs from H2O2-induced cell death. GB induced the phosphorylation of eNOS, Akt and p38, which in turn promoted cell proliferation and function. In conclusion, the present study demonstrates that GB, at a near medical applied dose, increases the number and functional activities of EPCs with involvement of Akt/endothelial nitric oxide synthase and mitogen-activated protein kinase (MAPK)/p38 signal pathways. These findings raise the intriguing possibility that GB may play an important role in the protection and revascularization of blood vessels.

Smoking and Endothelial Progenitor Cells: A Revision of Literature

Accumulating evidence indicates that circulating endothelial progenitor cells (EPCs) derived from bone marrow contribute to reendothelialization of injuried vessels as well as neo-vascularization of ischemic lesions in either a direct or an indirect way. Moreover, the number and/or the functional activity of EPCs are inversely correlated with risk factors for cardiovascular disease. Among the different risk factors, cigarette smoking is a major cause of reducing the numbers and function of circulating EPCs. This review is a revision of recent literature on EPC alteration associated with smoking. In particular, we show the recent observation on the effects of active and second hand smoke (SHS) exposure on EPC number and functional activity. This review also considers the effects of nicotine and other smoke compounds on EPC number and activity, in in vitro and in vivo models.

Abstract 4909: Nox2-Containing NADPH Oxidase Deficiency Protects Against Age-Dependent Inhibition of Neovascularization

Background: We have previously shown that aging is associated with an impaired angiogenic response following ischemia. Because aging leads to an increased generation of reactive oxygen species (ROS), here we investigated the role of Nox2-containing NADPH oxidase ROS generation for the modulation of neovascularization by aging. Methods: Hindlimb ischemia was surgically induced by femoral artery removal in young (5–7 weeks) and older (9 months) Nox2 −/− and control (Nox2 +/+ ) mice. Results: We found that blood flow recovery is significantly reduced in older compared to younger mice at day 21 after surgery (Doppler flow ratios: 0.51± 0.05 vs. 0.72± 0.05; p&lt;0.05). At the microvascular level, capillary density was also significantly reduced in ischemic muscles of older animals (53.4± 6.3 capillaries/field vs. 82.5± 4.9 capillaries/field; p&lt;0.001). This was associated with a significant increase of oxidative stress levels (nitrotyrosine immunostaining) in ischemic tissues of older animals. Importantly, we found that Nox2 deficiency reduces oxidative stress levels in ischemic tissues and restores blood flow recuperation in older animals. Endothelial progenitor cells (EPCs) have been shown to have an important role for postnatal neovascularization. Here we found that the functional activities of EPCs (adhesion to HUVECs, migration) are significantly impaired in old compared to young mice. However, Nox2 deficiency rescues EPCs functional activities in older animals. We also demonstrate an age-dependent pathological increase of oxidative stress levels (DCF-DA, DHE) in EPCs that is not present in Nox2-deficient animals. Conclusion: Nox2-containing NADPH oxidase deficiency protects against age-dependent impairment of neovascularization. Potential mechanisms include reduced ROS generation in ischemic tissues and preserved angiogenic activities of EPCs.

Sildenafil Increases Endothelial Progenitor Cell Function and Improves Ischemia-Induced Neovascularization in Hypercholesterolemic Apolipoprotein E–Deficient Mice

Hypercholesterolemia is associated with impaired neovascularization in response to ischemia. Potential mechanisms include defective NO bioactivity and a reduction in the number/function of endothelial progenitor cells (EPCs). Here we tested the hypothesis that sildenafil, a phosphodiesterase 5 inhibitor that increases NO-driven cGMP levels, could stimulate EPC function and improve ischemia-induced neovascularization in hypercholesterolemic conditions. Apolipoprotein E-deficient (ApoE(-/-)) mice were treated (or not treated) with sildenafil (40 mg/kg per day in water), and hindlimb ischemia was surgically induced by femoral artery removal. Sildenafil treatment led to an improved blood flow recovery, an increased capillary density, and a reduction of oxidative stress levels in ischemic muscles at day 7 after surgery. Sildenafil therapy is associated with an increased activation of angiogenic transduction pathways, including Akt, p44/42 mitogen-activated protein kinase, and p38. In vitro, sildenafil increases cellular migration and tubule formation of mature endothelial cells (human umbilical vascular endothelial cells) in a cGMP-dependent manner. In vivo, ApoE(-/-) mice treated with sildenafil exhibit a significant increase in the number of bone marrow-derived EPCs. Moreover, the angiogenic activities of EPCs (migration and adhesion) are significantly improved in ApoE(-/-) mice treated with sildenafil. In summary, this study demonstrates that sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic ApoE(-/-) mice. The mechanisms involve beneficial effects on angiogenic transduction pathways together with an increase in the number and the functional activity of EPCs. Sildenafil could constitute a novel therapeutic strategy to reduce tissue ischemia in atherosclerotic diseases.

Adiponectin levels are associated with the number and activity of circulating endothelial progenitor cells in patients with coronary artery disease

To study the relationship between plasma adiponectin concentration and the functional activities of circulating endothelial progenitor cells (EPCs) in patients with coronary artery disease (CAD). Circulating EPCs were enumerated as AC133(+)/KDR(+) cells via flow cytometry and identified by co-staining with DiI-acLDL and fluorescein isothiocyanate (FITC)-conjugated lectin under a fluorescent microscope. The migratory capacity of EPCs was measured by modified Boyden chamber assay. Adhesion capacity was performed to count adherent cells after replating EPCs on six-well culture dishes coated with fibronectin. The number of circulating EPCs (AC133(+)/KDR(+) cells) decreased significantly in CAD patients, compared with control subjects [(74.2+/-12.3) vs (83.5+/-12.9) cells/ml blood, P<0.01]. In addition, the number of EPCs also decreased in CAD patients after ex vivo cultivation [(54.4+/-8.6) vs (71.9+/-11.6) EPCs/field, P<0.01]. Both circulating EPCs and differentiated EPCs were positively correlated with plasma adiponectin concentration. The functional activities of EPCs from CAD patients, such as migratory and adherent capacities, were also impaired, compared with control subjects, and positively correlated with plasma adiponectin concentration. The study demonstrates that the impairment of the number and functional activities of EPCs in CAD patients is correlated with their lower plasma adiponectin concentrations.

Effect of L-arginine on circulating endothelial progenitor cells in hypercholesterolemic rabbits

The cardiovascular disease risk factors result in endothelial cells apoptosis and injury. Recently, endothelial progenitor cells (EPCs) have been shown to participate in ongoing endothelial repair. Nitric Oxide (NO) is essential for mobilization and functional activity of EPCs. We hypothesized that L-arginine (NO precursor) supplementation may increase EPCs number in hypercholesterolemic rabbits. EPCs, plasma levels of nitrite, von Willebrand Factor (vWF) and Intima-Media thickness ratio were determined in rabbits feeding high-cholesterol diet or high-cholesterol diet with L-arginine(3% in drinking water). L-arginine supplementation had a significant effect on the nitrite levels, and EPC numbers, and inhibited vWF increment and atherosclerosis progression ( p < 0.05). Our results suggest that the beneficial effect of L-arginine in prevention of atherosclerosis may be due to the modulation of EPC levels.

Changes in number and biological function of endothelial progenitor cells in hypertension disorder complicating pregnancy

To examine the changes in number and function of endothelial progenitor cells (EPCs) from peripheral blood (PB) in hypertension disorder complicating pregnancy (HDCP), 20 women with HDCP and 20 normal pregnant women at the third trimester were studied. Mononuclear cells (MNCs) from PB were isolated by Ficoll density gradient centrifugation. EPCs were identified by positive expression of both CD34 and CD133 under fluorescence microscope and positive expression of factor VIII as shown by immunocytochemistry. The number of EPCs was flow-cytometrically determined. Proliferation and migration of EPCs were measured by MTT assay and modified Boyden chamber assay, respectively. The adhesion activity of EPCs was detected by counting the number of the adherent cells. The results showed that, compared with normal pregnant women, the number of EPCs was significantly reduced in HDCP (4.29%+/-1.21% vs 15.32%+/-2.00%, P<0.01), the functional activity of EPCs in HDCP, such as proliferation (13.45%+/-1.68% vs 18.45%+/-1.67%), migration (37.25+/-7.28 cells/field vs 67.10+/-9.55 cells/field) and adhesion activity (20.65+/-5.19 cells/field vs 34.40+/-6.72 cells/filed) was impaired (P<0.01). It is concluded that the number and function of EPCs are significantly decreased in HDCP.

Pharmacological approaches to improve endothelial repair mechanisms

Endothelial injury is thought to play a pivotal role in the development and progression of vascular diseases, such as atherosclerosis, hypertension or restenosis, as well as their complications, including myocardial infarction or stroke. Accumulating evidence suggests that bone marrow-derived endothelial progenitor cells (EPCs) promote endothelial repair and contribute to ischemia-induced neovascularization. Coronary artery disease and its risk factors, such as diabetes, hypercholesterolemia, hypertension and smoking, are associated with a reduced number and impaired functional activity of circulating EPCs. Moreover, initial data suggest that reduced EPC levels are associated with endothelial dysfunction and an increased risk of cardiovascular events, compatible with the concept that impaired EPC-mediated vascular repair promotes progression of vascular disease. In this review we summarize recent data on the effects of pharmacological agents on mobilization and functional activity of EPCs. In particular, several experimental and clinical studies have suggested that statins, angiotensin-converting enzyme inhibitors, angiotensin II type 1 receptor blockers, PPAR-γ agonists and erythropoietin increase the number and functional activity of EPCs. The underlying mechanisms remain largely to be defined; however, they likely include activation of the PI3-kinase/Akt pathway and endothelial nitric oxide synthase, as well as inhibition of NAD(P)H oxidase activity of progenitor cells.

High glucose reduces cathepsin L activity and impairs invasion of circulating progenitor cells

Endothelial progenitor cells (EPC) significantly contribute to neovascularization and endothelial regeneration. Risk factors for coronary artery disease, particularly diabetes mellitus, reduce the number and functional activity of EPC. As we have recently shown, expression and activity of the matrix degrading cysteine protease cathepsin L in EPC is required for tissue invasion and EPC-mediated improvement of neovascularization. Therefore, we investigated the effect of high glucose and diabetes mellitus on EPC invasion and cathepsin L activity. Incubation of EPC with high levels of glucose (10–30 mM) dose-dependently decreased cathepsin L activity (glucose 20 mM: 67±4% compared to control; p<0.05) and protein expression (48±5% of control, p<0.05). In contrast, other proteases of the cathepsin family such as cathepsins D and O, and the matrix metalloproteinases MMP-2 and MMP-9 were not altered with high glucose. Cathepsin L mRNA was not affected suggesting that a posttranscriptional mechanism is responsible for cathepsin L down-regulation. As a functional consequence, high glucose significantly reduced the gelatinolytic activity and invasion of EPC (50±5% of control). Importantly, EPC of patients with type 2 diabetes revealed profoundly decreased cathepsin L expression and activity as compared to EPC derived from healthy controls. Taken together, high glucose significantly reduces the protein expression and activity of cathepsin L, which is involved in matrix degradation and required for invasion of EPC into the ischemic tissue, and, thereby, may limit the functional capacity of EPC to improve neovascularization in diabetics.

Effect of red wine antioxidants and minor polyphenolic constituents on endothelial progenitor cells after physical training in mice

Circulating endothelial progenitor cells (EPCs) play a significant role in regeneration of damaged blood vessels. Levels and functional activities of EPCs are noticeable altered by risk factors for coronary heart disease (CHD) and compounds that can prevent or ameliorate EPC dysfunction are currently of special clinical interest. Here, we evaluate the effects of red wine (RW) on EPCs in C57BL/6J mice subjected to physical exercise. FACS computed counting showed a significant increase of EPC number ( P < 0.05) in mice after short-term supplementation with RW. VEGF serum concentration was significantly increased by physical training in the presence or absence of RW supplementation ( P < 0.001). These in vivo observations support previous in vitro observation of the beneficial effect of RW in the modulation of EPC levels.

Abstract 461: Sildenafil Improves Ischemia-Induced Neovascularization in ApoE-Deficient Mice

Background: Sildenafil is widely used to treat erectile dysfunction. Sildenafil inhibits phospho-diesterase 5 (PDE5), thereby increasing cGMP levels in target tissues. The NO-cGMP pathway has been shown to be essential for angiogenesis, vasculogenesis and postnatal neovascularization. Therefore, here we tested the hypothesis that sildenafil might improve neovascularization following ischemia. Methods and results: Hypercholesterolemic ApoE−/− mice, which are characterised by reduced neovascularization in response to ischemia, were treated or not with sildenafil (40 mg/kg/day in water) for the whole duration of the study. Sildenafil was well tolerated by ApoE−/− mice and led to a significant increase of plasmatic cGMP levels compared to controls (13.4±1.9 vs. 4.6±2.6 pmol/mL, p&lt;0.05). After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. Sildenafil treatment led to a significantly faster rate of blood flow recovery, as assessed by Laser Doppler measurement at day 7 after surgery (55.5% increase, p&lt;0.05). We found that sildenafil treatment is associated with an increased activation of angiogenic transduction pathways such as Akt, p44/42 MAPK, and p38 in ischemic muscles (phosphospecific Western blots). Moreover, sildenafil treatment leads to a significant reduction of oxidative stress in ischemic tissues (nitrotyrosine staining). Endothelial progenitor cells (EPCs) have been shown to participate to postnatal neovascularization. We found that ApoE−/− mice treated with sildenafil have a significant increase in the number of bone marrow EPCs (DiI-AcLDL and FITC-Lectin staining) compared to untreated mice (272±18 vs. 187±17 cells per field, p&lt;0.05). Moreover, the adhesion (19.7±2.1 vs. 10.0±1.9 EPCs per field, p&lt;0.05) and the migratory capacity (28.5±1.8 vs. 15.2±1.3 EPCs per field, p&lt;0.0001) of EPCs are significantly improved in ApoE−/− mice treated with sildenafil. Conclusions: Sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic conditions. The mechanisms involve beneficial effects of sildenafil on angiogenic pathways in ischemic tissues together with an increase in the number and the functional activity of EPCs.

Moderate consumption of red wine (cabernet sauvignon) improves ischemia‐induced neovascularization in ApoE‐deficient mice: effect on endothelial progenitor cells and nitric oxide

Moderate consumption of red wine is associated with a decreased incidence of cardiovascular diseases in populations with relatively high amount of fat in the diet. However, the mechanisms involved in this protective effect are not completely understood. Here we show that moderate consumption of red wine (equivalent to 2 glasses/day in humans) but not ethanol only, improves blood flow recovery by 32% after hindlimb ischemia in hypercholesterolemic ApoE-deficient mice. In ischemic tissues, red wine consumption reduces oxidative stress and increases capillary density by 46%. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascularization. We found that the number of EPCs is increased by 60% in ApoE mice exposed to red wine. Moreover, the migratory capacity of EPCs is significantly improved in red wine-drinking mice. The wine used in our study is a cabernet sauvignon from Languedoc-Roussillon, France, which contains a relatively high concentration (4-6 mg/L) of the polyphenolic antioxidant resveratrol. We demonstrate that resveratrol can rescue oxidized low-density lipoprotein (oxLDL)-induced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Resveratrol exposure is also associated with increased activation of Akt/eNOS together with a restoration of nitric oxide production in HUVECs exposed to oxLDL. Our study suggests that moderate consumption of red wine improves ischemia-induced neovascularization in high-cholesterol conditions by increasing the number and the functional activities of EPCs and by restoring the Akt-eNOS-NO pathway.

Antioxidant protection: yet another function of endothelial progenitor cells?

Previously considered purely as a mechanical monolayer barrier, the modern view of the endothelium is of an extremely active ‘biochemical laboratory’ acting in both an endocrine and paracrine manner to maintain and regulate vascular health. The strong association between endothelial damage/dysfunction and cardiovascular disease has been the subject of intense research. Indeed, the endothelium is believed to play a pivotal role in many cardiovascular disease states, including hypertension. However, the concept of ‘endothelial dysfunction’ should not be limited purely to endothelial abnormalities, but should include consideration of a host of anomalies that occur in the conduit vessel wall, within the blood and perhaps even extended to alterations in the number and/or function of endothelial progenitor cells (EPCs) within the bone marrow. In all individuals, the vascular wall and the endothelium undergo a constant process of injury and repair in response to both mechanical and chemical injury. Accumulating evidence suggests that circulating EPCs derived from the bone marrow play an important role in the maintenance of vascular integrity by augmentation of neovascularization, endothelial repair and promotion of endothelial function. Both physiological (e.g. aging) and pathological (e.g. hypercholesterolemia, diabetes mellitus and coronary artery disease) conditions are associated with decreased number of circulating EPCs. Transplantation of EPCs from healthy individuals has suggested that these cells have capacity for potent angiogenic properties and may promote preservation of ischemic tissue. However, some disparity exists between the pronounced angiogenic effects of EPCs on animal models and the rather modest effects seen in clinical trials on human subjects. Importantly, the data would appear to imply that the number of available progenitors is not the primary factor for successful cellular revascularization, but instead the functional capacity of human EPCs is of paramount importance. Impaired EPC functionality in patients with cardiovascular risk factors is well documented. Of note, an inverse correlation is found between the number and functional activity of EPCs and cardiovascular risk factors among apparently healthy persons and also in patients with coronary artery disease. Furthermore, the number of EPCs has been demonstrated to correlate with endothelial function and is a better predictor for future cardiovascular risk than the combined Framingham risk factor score. The intimate mechanisms responsible for restoration of endothelial function remain unclear and this has prompted researchers to study different aspects of EPC biology and function. Among the large number of vasoactive substances produced by endothelial cells, nitric oxide (NO) appears to be of crucial importance. Some have speculated that endothelial NO activity is the hallmark of endothelial function, and the release of NO plays a critical role in the control of vascular tone. Potent antiaggregatory and proangiogenic properties of NO are also well determined. The clinical implications of endothelial dysfunction and the association between reduced NO bioavailability, cardiovascular risk factors (including hypertension) and cardiac events is well established. Moreover, NO production by EPCs has been shown to be not only marker of cellular maturation but also a factor which has a strong impact on their proangiogenic properties. How can we improve endothelial function? Apart from pharmacological methods, physical exercise has been associated with a significant reduction in cardiovascular morbidity and mortality, and this may be mediated through exercise-related improvements in endothelial function. Shear stress is one of the most effective endothelial stimulators, and results in an upregulation of the endothelial NO synthase activity and NO production, at least in an experimental model. The beneficial effect of exercise training on endothelium-dependent vasodilatation observed in several Correspondence: Professor GYH Lip, Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Dudley Road, Birmingham B18 7QH, UK. E-mail: g.y.h.lip@bham.ac.uk Published online 22 February 2007 Journal of Human Hypertension (2007) 21, 343–346 & 2007 Nature Publishing Group All rights reserved 0950-9240/07 $30.00