Recruitment Of Endothelial Cells Research Articles

What are chemokine signalling systems doing in the brain?

What are chemokine receptors and ligands doing in the brain? There are 40–50 chemokine ligands, small secreted proteins of 60–100 amino acids. About 20 chemokine receptors exist, ordered in distinct families and coupled to G-proteins. One might think these signalling molecules, which act to attract migrating cells, have a uniquely pathological role in neurodegenerative diseases or to mediate inflammatory responses. Their functions however, seem likely to be wider than that (Rostene et al. 2007; Guyon et al. 2008). Certainly, chemokine signalling contributes to inflammatory responses by attracting cells of the immune system to regions of infection or damage in the brain. But chemokines are also involved in other processes involving cell motility including the control of neuronal migration during brain development (Tiveron & Cremer, 2008) and also the recruitment of endothelial progenitor cells to form blood vessels during cerebral angiogenesis. Chemokine ligands are liberated by microglia, by astrocytes and also by some neurones. The ligand CXCL12, for instance, is present in synaptic vesicles of granule cells of the hippocampal dentate gyrus. Both glial and neuronal cells express G-protein coupled, cytokine receptors. Often they are expressed selectively in distinct brain areas by specific types of neurones, sometimes with a distinct developmental profile. For instance, adult dopaminergic cells of the substantia nigra express the cytokine receptor CCR2, while cortical Cajal-Retzius cells, which largely disappear during development, express the CXCR4 receptor. But what are they doing in the brain? One way to find out is to study the physiology of cells that express chemokine receptors. Such work has been greatly facilitated by the development of BAC transgenic animals in which fluorescent reporter proteins reflect the expression of specific receptor molecules. In a recent report in The Journal of Physiology, Marchionni and colleagues (2010) describe work on a CXCR4-EGFP animal. This chemokine receptor binds a specific chemokine ligand, CXCL12, also known as the stromal-cell derived factor 1. The CXCR4 receptor is expressed by progenitor cells in the dentate gyrus of post-natal hippocampus (Bhattacharyya et al. 2008). Marchionni et al. now show that Cajal-Retzius cells in the CA1 region of the hippocampus also express the receptor. Cajal-Retzius cells secrete the large glycoprotein reelin, which has a crucial role in the migration of cortical neurones. In the neocortex, they seem to be largely transient, disappearing or at least stopping reelin secretion, when migration is complete. In the CA1 region of the hippocampus however, Marchionni and colleagues show that the Cajal-Retzius cells remain at least until P16–P24 when their experiments were performed. The somata of fluorescent cells in CXCR4-EGFP animals were found in the stratum lacunosum-moleculare of the CA1 region, so they are not pyramidal cells. They express reelin as expected, but also the Ca2+-binding protein calretinin, a marker of subsets of cortical interneurones. Are these chemokine receptor-expressing cells interneurones, then? Electrophysiological and anatomical arguments suggest probably not. Marchionni et al. show their electrical properties differ markedly from typical GABAergic interneurons of the same layer. Furthermore, electron microscopy revealed that axons of these Cajal-Retzius cells form terminals which, while containing transmitter vesicles, do not face classical postsynaptic specializations. And yet, both these points could be otherwise explained, so conclusive proof of the identity of released transmitter awaits a simultaneous record from a postsynaptic cell. If the output of the reelin and CXCR4-expressing fluorescent cells is not completely resolved, they are clearly integrated into synaptic circuits of the CA1 region. They receive sparse GABAergic synaptic events, which can trigger action potentials, due presumably to high levels of internal chloride. However, they express few, if any, glutamatergic receptors. These cells should of course respond to the CXRC4 ligand, stromal-cell derived factor 1 or CXCL12. And they do. It generates a slow probably K+ current mediated hyperpolarization. So Marchionni and colleagues have set the stage to ask in a much more informed way the question: what is this chemokine signalling system doing? CXRC4 receptors are expressed by adult Cajal-Retzius cells integrated into circuits in the CA1 region of the hippocampus. Probably, at P12–P24 in the CA1 region, they are not participating in migration. Possibly, they reflect a strategic signalling reserve that could be rapidly expanded when inflammatory processes are engaged. More probably, perhaps they participate in signalling in the adult CA1 region. The article shows that activation of CXRC4 receptors by their ligand CXCL12 abolishes the spontaneous discharges of these Cajal-Retzius cells. So chemokine signalling should abolish release of molecules contained in the vesicles of Cajal-Retzius cell axons. The absence of classical postsynaptic structures and the presence in the stratum lacunosum-moleculare of CA1 points to effects by volume transmission on afferents terminating in this layer. It is intriguing to wonder whether that volume transmission is mediated by GABA, or glutamate or even reelin. Whichever it is, this article seems likely to modify once again our vision of hippocampal circuit function.

Expression of stem cell factor/c‐kit signaling pathway components in diabetic fibrovascular epiretinal membranes

Abstract Purpose Stem cell factor (SCF)/c‐kit signaling promotes recruitment of endothelial progenitor cells and contributes to ischemia‐induced new vessel formation. We investigated the expression of the components of this pathway including c‐kit, SCF, granulocyte colony‐stimulating factor (G‐CSF), endothelial nitric oxide synthase (eNOS), and the chemokine receptor CXCR4 in proliferative diabetic retinopathy (PDR) epiretinal membranes. Methods Membranes from 8 patients with active PDR and 12 patients with inactive PDR were studied by immunohistochemistry. Results Blood vessels expressed c‐kit, SCF, G‐CSF, eNOS, and CXCR4 in 18, 15, 19, 20 and 20 out of 20 membranes, respectively. Significant correlations were detected between number of blood vessels expressing CD34 and number of blood vessels expressing SCF (r=0.463; p=0.04), G‐CSF (r=0.87; p<0.001), eNOS (r=0.864; p<0.001), and CXCR4 (r=0.864; p<0.001). Stromal cells expressed c‐kit, SCF, eNOS, and CXCR4 in 19, 15, 20, and 20 membranes, respectively. The numbers of blood vessels expressing CD34 (p=0.005), c‐kit (p=0.03), G‐CSF (p=0.007), eNOS (p=0.001), and CXCR4 (p=0.018), and stromal cells expressing c‐kit (p=0.013), SCF (p<0.001), eNOS (p=0.048), and CXCR4 (p=0.003) were significantly higher in active membranes than in inactive membranes. Conclusion SCF/c‐kit signaling might contribute to neovascularization in PDR.

The PI3K/Akt pathway upregulates Id1 and integrin α4 to enhance recruitment of human ovarian cancer endothelial progenitor cells

BackgroundEndothelial progenitor cells (EPCs) contribute to tumor angiogenesis and growth. We aimed to determine whether inhibitors of differentiation 1 (Id1) were expressed in circulating EPCs of patients with ovarian cancer, whether Id1 could mediate EPCs mobilization and recruitment, and, if so, what underlying signaling pathway it used.MethodsCirculating EPCs cultures were from 25 patients with ovarian cancer and 20 healthy control subjects. Id1 and integrin α4 expression were analyzed by real-time reverse transcription-polymerase chain reaction and western blot. EPCs proliferation, migration, and adhesion were detected by MTT, transwell chamber, and EPCs-matrigel adhesion assays. Double-stranded DNA containing the interference sequences were synthesized according to the structure of a pGCSIL-GFP viral vector and then inserted into a linearized vector. Positive clones were identified as lentiviral vectors that expressed human Id1 short hairpin RNA (shRNA).ResultsId1 and integrin α4 expression were increased in EPCs freshly isolated from ovarian cancer patients compared to those obtained from healthy subjects. siRNA-mediated Id1 downregulation substantially reduced EPCs function and integrin α4 expression. Importantly, Inhibition of PI3K/Akt inhibited Id1 and integrin α4 expression, resulting in the decreasing biological function of EPCs.ConclusionsId1 induced EPCs mobilization and recruitment is mediated chiefly by the PI3K/Akt signaling pathway and is associated with activation of integrin α4.

Mechanism of endothelial progenitor cell recruitment into neo-vessels in adjacent non-tumor tissues in hepatocellular carcinoma

BackgroundWe investigated the distribution and clinical significance of mobilized endothelial progenitor cells (EPCs) in hepatocellular carcinoma (HCC). We found that many more EPCs were recruited to nonmalignant liver tissue (especially into adjacent non-tumor tissues (AT)) than to tumor vessels. These results suggest that the mechanism underlying the recruitment of EPCs into microvessels in AT merits further investigationMethodsAngiogenic factors were detected in three tissue microarrays comprising normal liver, paired tumor tissue (TT) and AT from 105 patients (who had undergone hepatectomy for HCC) using immunohistochemistry. Also, the number of EPCs (positive for Sca-1, Flk-1 and c-Kit) in the blood and liver of cirrhotic mice were determined by flow cytometry and immunohistochemistry. The distribution of these labeled EPCs in tumor and non-tumor tissues was then studied.ResultsThe results from the tissue microarrays showed that the expression levels of VEGF-A, bFGF, TGF-β, MCP-1, TSP-1, MMP-9, TIMP-2, and endostatin were significantly higher in AT than in either normal liver or TT (p < 0.05), but no significant difference was found in the expression levels of COX-2 and NOS-2 between AT and TT. The expression of VEGF-A, bFGF, TGF-β, MCP-1, TSP-1, MMP-9, TIMP-2, endostatin, COX-2, and NOS-2 in normal liver tissue was weaker than that in AT or TT. In cirrhotic mice, the number of circulating endothelial progenitor cells gradually increased, before decreasing again. In this mouse model, increased numbers of EPCs were recruited and homed specifically to the cirrhotic liver.ConclusionsBoth liver cirrhosis and HCC led to increased expression of pro-angiogenic factors, which resulted in the recruitment of EPCs into AT. Also, EPCs were mobilized, recruited and homed to cirrhotic liver. The unique pathology of HCC coupled with liver cirrhosis may, therefore, be associated with the distribution and function of EPCs.

Oxygen/ozone protects the heart from acute myocardial infarction through local increase of eNOS activity and endothelial progenitor cells recruitment

The purpose of this study is to investigate whether an oxygen/ozone (O(2)/O(3)) mixture protects the heart from acute myocardial infarction through local involvement of endothelial nitric oxide synthase (eNOS) and endothelial progenitor cells (EPCs). Male Sprague-Dawley rats were subject to 25-min occlusion and 2-h reperfusion of the left descending coronary artery. O(2)/O(3) mixture was insufflated i.p. 30 min prior to ischemia/reperfusion (I/R) procedure at doses of 100, 150, and 300 microg/kg. Myocardial infarct size (IS) measurement and myocardial immunohistochemistry for EPCs were done. For these latter cells, immunoreactivities for CD34, and CD117/c-kit were assessed within the infarcted tissue. Moreover, cardiac eNOS was monitored. I/R in rats treated with O(2) produced an IS as a percentage of the area at risk (IS/AR) equal to 51 +/- 5%. I/R in rats treated with the O(2)/O(3) mixture showed reduced IS (for example, IS/AR for 150 microg/kg O(2)/O(3) was 35 +/- 2.1%; P < 0.01 vs. O(2)). The O(2)/O(3) cardio-protection was paralleled by an increased number of immunopositive particles per area for CD34 and CD117/c-kit. The increase of these markers was associated with an increase of cardiac eNOS expression as assayed by immunohistochemistry. Interestingly, N5-(1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO), a selective eNOS activity inhibitor (30 mg/kg s.c.), prior to O(2)/O(3) and I/R almost abolished the cardio-protection exerted by the O(2)/O(3) mixture. L-NIO also abolished the increase in immunostaining for CD-34 and CD117/c-kit as compared with the rats receiving O(2)/O(3) and I/R only. O(2)/O(3) mixture protects the heart from acute myocardial infarction through local increase of eNOS expression/activity and consequent EPCs recruitment.

Sequential delivery of vascular endothelial growth factor and sphingosine 1-phosphate for angiogenesis

Angiogenesis is an organized series of events, beginning with vessel destabilization, followed by endothelial cell re-organization, and ending with vessel maturation. Vascular endothelial growth factor (VEGF) aids in vascular permeability and endothelial cell recruitment while sphingosine 1-phosphate (S1P) stimulates vascular stability. Accordingly, VEGF may inhibit vessel stabilization while S1P may inhibit endothelial cell recruitment. For this reason, we created a new externally-regulated delivery model that not only permits sustained release of bioactive factors, but also temporal separation of the delivery of growth factors. Using this model, sequential delivery of factors was first confirmed in vitro with associated endothelial cells responding in a dose dependent manner. Furthermore, using a modified murine Matrigel plug model, it is apparent that delivery strategies where VEGF presentation is temporally separated from S1P presentation not only led to greater recruitment of endothelial cells, but also higher maturation index of associated vessels.

Recruitment of human endothelial cells expressing alpha-gal epitopes into tumor vessels as a novel strategy to promote complement dependent tumor regression

Recruitment of human endothelial cells expressing alpha-gal epitopes into tumor vessels as a novel strategy to promote complement dependent tumor regression

Investigation of vasculogenic mimicry in sebaceous carcinoma of the eyelid

Vasculogenic mimicry (VM) is a newly proposed pattern of tumour angiogenesis that has been identified in some malignancies and is associated with poor prognosis. The purpose of this study was to investigate whether sebaceous carcinomas of the eyelid exhibit VM and to determine whether these fluid-conducting patterns are associated with clinicopathologic features, the number of microvessels and the levels of endothelial growth factor (VEGF) and matrix metalloprotease-2 (MMP-2) in tumours. Forty paraffin-embedded samples of sebaceous carcinoma of the eyelid were collected, along with complete clinical and pathologic data for all the cases. Tissue sections were stained for CD34, periodic acid and Schiff (PAS), VEGF and MMP-2. VM was identified by the presence of PAS-positive and CD34-negative loops lined by tumour cells. The VM status of tumour samples was compared with the clinical and pathological data using statistical tests. The levels of VEGF, MMP-2 and the number of microvessels were compared between patients with and without VM. VM was detected in 14 of 40 (35%) tumour samples. The existence of VM in tumours was associated with tumour size (p=0.007) and recurrence (p=0.021). The number of microvessels was lower in tumours with VM (13.03+/-4.02 versus 22.99+/-7.72; p<0.0001). The staining index of MMP-2 was higher in tumours with VM (27.43, range: 0-5.3) compared to tumours without VM (16.77, range: 0-2.7; p=0.004). However, there was no difference in the expression of VEGF between groups with and without VM (p=0.244). Vasculogenic mimicry is present in sebaceous carcinoma of the eyelid making it an unfavourable prognosis sign. MMP-2 is associated with VM formation in sebaceous carcinoma of the eyelid.

Cilostazol enhances neovascularization in the mouse hippocampus after transient forebrain ischemia

Cilostazol is known to be a specific type III phosphodiesterase inhibitor, which promotes increased intracellular cAMP levels. We assessed the effect of cilostazol on production of angioneurins and chemokines and recruitment of new endothelial cells for vasculogenesis in a mouse model of transient forebrain ischemia. Pyramidal cell loss was prominently evident 3-28 days postischemia, which was markedly ameliorated by cilostazol treatment. Expression of angioneurins, including endothelial nitric oxide synthase, vascular endothelial growth factor, and brain-derived neurotrophic factor, was up-regulated by cilostazol treatment in the postischemic hippocampus. Cilostazol also increased Sca-1/vascular endothelial growth factor receptor-2 positive cells in the bone marrow and circulating peripheral blood and the number of stromal cell-derived factor-1alpha-positive cells in the molecular layer of the hippocampus, which colocalized with CD31. CXCR4 chemokine receptors were up-regulated by cilostazol in mouse bone marrow-derived endothelial progenitor cells, suggesting that cilostazol may be important in targeting or homing in of bone marrow-derived stem cells to areas of injured tissues. CD31-positive cells were colocalized with almost all bromodeoxyuridine-positive cells in the molecular layer, indicating stimulation of endothelial cell proliferation by cilostazol. These data suggest that cilostazol markedly enhances neovascularization in the hippocampus CA1 area in a mouse model of transient forebrain ischemia, providing a beneficial interface in which both bone marrow-derived endothelial progenitor cells and angioneurins influence neurogenesis in injured tissue. (c) 2010 Wiley-Liss, Inc.

Recent concepts of antiangiogenic therapy

In 2004, a randomized, controlled phase III clinical trial showed that the addition of bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF)-A, to conventional chemotherapy prolonged the survival of patients with metastatic colorectal cancer. A number of clinical trials are presently underway to test the utility of several angiogenic inhibitors against a variety of malignancies. The original concept of antiangiogenic therapy was the inhibition of outgrowth of new blood vessels; however, it soon became evident that bevacizumab could affect the vasculature through various mechanisms. Recent studies have shown that antiangiogenic agents can normalize the tumor vasculature and prevent the recruitment of endothelial progenitor cells from the bone marrow. Some preclinical studies have also shown that antiangiogenic agents prevent metastasis by modulating the premetastatic niche. Understanding these detailed mechanisms provides the rationale for combination therapy using antiangiogenic agents and cytotoxic chemotherapy, and will lead to more effective treatment strategies. In this review, we summarize the present understanding of the mechanisms of action of antiangiogenic agents and discuss the future prospects of antiangiogenic therapies.

Endothelial Progenitor Cells Homing and Renal Repair in Experimental Renovascular Disease

Tissue injury triggers reparative processes that often involve endothelial progenitor cells (EPCs) recruitment. We hypothesized that atherosclerotic renal artery stenosis (ARAS) activates homing signals that would be detectable in both the kidney and EPCs, and attenuated on renal repair using selective cell-based therapy. Pigs were treated with intrarenal autologous EPC after 6 weeks of ARAS. Four weeks later, expression of homing-related signals in EPC and kidney, single kidney function, microvascular (MV) density, and morphology were compared with untreated ARAS and normal control pigs (n = 7 each). Compared with normal EPC, EPC from ARAS pigs showed increased stromal cell-derived factor (SDF)-1, angiopoietin-1, Tie-2, and c-kit expression, but downregulation of erythropoietin (EPO) and its receptor. The ARAS kidney released the c-kit-ligand stem cell factor, uric acid, and EPO, and upregulated integrin beta2, suggesting activation of corresponding homing signaling. However, angiopoietin-1 and SDF-1/CXCR4 were not elevated. Administration of EPC into the stenotic kidney restored angiogenic activity, improved MV density, renal hemodynamics and function, decreased fibrosis and oxidative stress, and attenuated endogenous injury signals. The ARAS kidney releases specific homing signals corresponding to cognate receptors expressed by EPC. EPC show plasticity for organ-specific recruitment strategies, which are upregulated in early atherosclerosis. EPC are renoprotective as they attenuated renal dysfunction and damage in chronic ARAS, and consequently decreased the injury signals. Importantly, manipulation of homing signals may potentially allow therapeutic opportunities to increase endogenous EPC recruitment.

Acrolein Inhalation Suppresses Recruitment and Mobilization of Endothelial Progenitor Cells

Exposure to cigarette smoke decreases the level of endothelial progenitor cells (EPCs) in peripheral blood (PB). We investigated the cardiovascular effects of inhaled acrolein, an unsaturated aldehyde present in high levels in tobacco smoke (up to 150 ppm). Adult male C57BL/6 mice were exposed to room air or acrolein (0.5, 1 or 5 ppm) for 2h, 6h, 1d, 2d or 4d (6h/d) and the EPC level in PB was measured by flow cytometry. To examine the proliferative capacity of EPCs, bone marrow cells (BMCs) were cultured on fibronectin‐coated slides. Brief exposure to acrolein (5 ppm, 6h) or 4 day (1 ppm) exposure led to a 42% decrease in PB Flk+/Sca+cells (air‐exposed: 8.9±1.7/μl, n=4; acrolein‐exposed: 4.9±0.5/μl, n=4, p&lt;0.05). BMCs isolated from mice exposed to acrolein for 1d (5 ppm) or 4d (1 ppm) formed more colonies than BMCs of air‐exposed mice (air‐exposed: 7±1 colonies; acrolein 14±1 colonies at 1d in culture, p&lt;0.001; air‐exposed 3±0.47 colonies; acrolein‐exposed 9±2 colonies after 4 days in culture, p&lt;0.001). Combined VEGF‐A (100 μg/kg, ip, 4d) and AMD3100 (5 mg/kg; ip, last day only) treatment increased PB Flk+/Sca+ cells (air‐exposed: 5.7/μ1±1.8, n=4; acrolein‐exposed: 11.06/μl±2.2, n=4, p&lt;0.05) in air‐exposed mice but not in acrolein‐exposed mice (1 ppm, 4d). These data indicate that PB EPCs are a sensitive target of airborne pollutants and that acrolein at concentrations encountered in tobacco smoke suppresses PB EPC level yet increases the proliferative capacity of BMCs. Acrolein‐induced depletion of circulating EPCs appears in part to be due to a defect in EPC mobilization and recruitment.COBRA: P20RR024489; PPG: P01ES011860

On the developmental origin of hepatic sinusoidal endothelium: confirmation of angiocirculatory vasculogenesis

Developmental mechanisms reported to account for the origin of hepatic sinusoidal endothelium include (1) in situ differentiation of mesenchymal cells (Sherer, 1991), (2) sprouting from preexisting vessels (Gouysse et al., 2002), (3) transdifferentiation of mesothelial cells (Pérez‐Pomares et al., 2004), and (4) recruitment of angioblasts and/or vascular endothelial cells (VECs) from the circulation (Pardanaud &amp; Eichmann, 2006; Zhang et al., 2006). The present study was designed to investigate whether the last of these mechanisms, here termed angiocirculatory vasculogenesis, plays a role in the development of hepatic sinusoidal endothelium. Chick livers were harvested from quail/chick parabioses maintained either in ovo or in shell‐less culture and were examined for evidence of quail VECs by immunostaining with QH1 antibody. Numerous positively‐stained cells were found to contribute to the sinusoids of parabiotic chick livers. Non‐parabiotic chick liver, serving as a negative control, lacked staining. These findings confirm that at least some of the cells that form these capillary‐level vascular structures are, as previously reported, acquired from circulating blood.Grant Funding Source: None

Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment

Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 microg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase (P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 microM), an inhibitor of MEK, which is a kinase upstream from Erk (P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity (P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation (P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.

Usefulness of Clopidogrel to Protect Against Diabetes-Induced Vascular Damage

Clopidogrel enhances the levels of endothelial nitric oxide and prostacyclin in tissue culture. We have previously described a marked increase in circulating endothelial cells (CECs), an ex vivo indicator of vascular injury, in patients with type 2 diabetes mellitus. We hypothesized that clopidogrel treatment would result in a decrease in CEC number and increased activity of endothelial progenitor cell recruitment signaling pathways in diabetic patients. CECs were isolated from the peripheral blood of 9 patients with type 2 diabetes using anti-CD146-coated Dynabeads. The cells were stained with acridine orange and counted by fluorescence microscopy. Endothelial progenitor cells were isolated in a similar fashion using anti-CD34 and anti-CD133 and assayed for expression of phosphorylated Akt and phosphorylated adenosine monophosphate kinase. The patients were then treated with clopidogrel 75 mg/day for 30 days, after which repeat blood specimens were analyzed. As previously observed, diabetic patients had an elevated number of CECs (mean 79 +/- 15 cells/ml peripheral blood), which was reduced by clopidogrel treatment (mean 10 +/- 4 cells/ml; p <0.001). This was associated with a significant increase in the expression of both phosphorylated Akt and phosphorylated adenosine monophosphate kinase (p </=0.05). In conclusion, clopidogrel reduces endothelial cell sloughing and increases expression of endothelial progenitor cell phosphorylated Akt and phosphorylated adenosine monophosphate kinase in the peripheral blood of patients with type 2 diabetes mellitus. This represents a novel mechanism by which this agent can promote improved vascular function, protect against oxidative stress, inhibit apoptosis, and attenuate vascular damage in patients with diabetes mellitus.

Exercise acutely reverses dysfunction of circulating angiogenic cells in chronic heart failure

Recruitment of endothelial progenitor cells (EPCs) and enhanced activity of circulating angiogenic cells (CACs) might explain the benefits of exercise training in reversing endothelial dysfunction in chronic heart failure (CHF) patients. We studied baseline EPC numbers and CAC function and the effect of a single exercise bout. Forty-one CHF patients (mild, n = 22; severe, n = 19) and 13 healthy subjects were included. Migratory activity of CACs was evaluated in vitro and circulating CD34+ and CD34+/KDR+ (EPC) cells were quantified by flow cytometry before and after cardiopulmonary exercise testing (CPET). Circulating stromal cell-derived factor-1alpha (SDF-1alpha) and vascular endothelial growth factor (VEGF) concentrations were measured. Both CAC migration as well as CD34+ cell numbers were significantly reduced in CHF, whereas CD34+/KDR+ cells were not different from controls. Endothelial dysfunction was related to impaired CAC migration (r = 0.318, P = 0.023). Cardiopulmonary exercise testing improved CAC migration in severe (+52%, P < 0.005) and mild CHF (+31%, P < 0.005), restoring it to levels similar to controls. Following CPET, SDF-1alpha increased in healthy controls and mild CHF (P < 0.005). Vascular endothelial growth factor, CD34+, and CD34+/KDR+ cell numbers remained unchanged. The present findings reveal a potent stimulus of acute exercise to reverse CAC dysfunction in CHF patients with endothelial dysfunction.

Hypoxia-induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non-cognate ligand of the CXC-family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine-like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC-derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia-induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non-classical mechanism and originated from pre-formed MIF stores. Early hypoxia-triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia-inducible factor (HIF)-1α-induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF-1α-induced de novo synthesis of MIF. To functionally investigate the role of hypoxia-inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL+ KDR+ CD31+ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose-dependent bell-shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia-conditioned HUVECs, an effect that was completely abrogated by anti-MIF- or anti-CXCR4-antibodies. Thus, hypoxia-induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia-induced myocardial damage.

Tumor angiogenesis and novel antiangiogenic strategies

Angiogenesis is essential for the development and growth of tumors. It is a highly regulated process that requires cross-talk between signaling pathways at all stages of blood vessel development and tumor growth, from the recruitment of endothelial cells to vessel maturation. This review summarizes tumor angiogenesis and describes the key signaling pathways governing blood vessel development. The role of angiogenesis in various tumor types is discussed, but the focus is on invasive breast cancer, a disease that will affect approximately 182,000 women in the USA in 2008. Research efforts over the past decade have identified numerous potential, as well as proven therapies with activity in breast cancer. These include chemotherapeutics as well as therapies that inhibit specific angiogenic pathways known as targeted agents. Some of the data from single- and multitargeted antiangiogenic agents are described in this review. "Published 2008 Wiley-Liss, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America."

Stromal Derived Factor-1α-Induced Recruitment of Endothelial Progenitor Cells Is Mediated by E-Selectin

Stromal Derived Factor-1α-Induced Recruitment of Endothelial Progenitor Cells Is Mediated by E-Selectin

The Role and Therapeutic Potential of Endothelial Progenitor Cells in Tumor Neovascularization

Although the cellular and molecular mechanisms of tumor growth and metastasis are not completely understood, it is established that formation and growth of new blood vessels is a conditio sine qua non for tumor survival, growth, and expansion. Numerous studies over the past decades demonstrated that neovascularization associated with tumor growth occurs via angiogenic and vasculogenic mechanisms that involve sprouting angiogenesis, intussusceptive angiogenesis, vessel co-option, vasculogenic mimicry, lymphangiogenesis, and the recruitment of endothelial progenitor cells (EPCs). Due to their ability to self-renew, circulate, home to the ischemic sites, and differentiate into mature endothelial cells, EPCs hold enormous potential to be used as a diagnostic and/or therapeutic agent in antitumor therapies. Hence, this review focuses on EPCs and their role in tumor angiogenesis with the emphasis on EPC recruitment/migration, and the potential use of EPCs as a therapeutic tool and imaging probe.