Proangiogenic Progenitor Cells Research Articles

Glomerular filtration rate predicts pro-vascular progenitor cell depletion: insights from the IPE-PREVENTION and ORIGINS-RCE studies

Abstract Background People living with cardiometabolic disease and impaired renal function are at increased risk of experiencing adverse cardiovascular outcomes(1,2). Recent evidence indicates that individuals living with atherosclerotic cardiovascular disease (ASCVD) and type 2 diabetes (T2D) exhibit a reduction in circulating vascular regenerative (VR) cells that are essential for maintaining blood vessel homeostasis(3). However, there is limited data on whether VR cell content changes in response to cardiometabolic and kidney disorders. Purpose We sought to determine if there is an association between estimated glomerular filtration rate (eGFR) and VR cell content in individuals with ASCVD and/or diabetes. Data were from participants enrolled in the IPE-PREVENTION and ORIGINS-RCE studies. Methods IPE-PREVENTION participants had moderately elevated triglycerides (1.5 - 5.65 mmol/L) with established ASCVD and/or T2D. ORIGINS-RCE participants had established ASCVD or T2D and at least one additional cardiovascular risk factor. Circulating mononuclear cells were isolated from peripheral blood and characterized using side scatter properties (SSC) and high aldehyde dehydrogenase activity to quantify pro-angiogenic early myeloid progenitor cells (ALDHhi), and primitive versus lineage-specific cell surface marker co-expression. Participants were stratified according to those with an eGFR <60 mL/min/1.73m², or ≥60 mL/min/1.73m². Results Median [IQR] eGFR of the pooled cohort (n=169) was 75 [59, 88] mL/min/1.73m²; 45% had an eGFR that was <60 mL/min/1.73m². The stratum with eGFR <60 mL/min/1.73m² had fewer circulating ALDHhiSSClow progenitor cells (0.037 vs. 0.051%; P<0.01) (Figure 1A) and ALDHhiSSClow cells expressing CD34+ and CD133+ (49 vs. 54%; P=0.03) (Figure 1B) than that with an eGFR ≥60 mL/min/1.73m². The predictive value of eGFR on ALDHhiSSClowCD34+CD133+ frequency was conserved even after controlling for age, sex, LDL-C, BMI, and HbA1c (P<0.01) (Table 1). The frequency of ALDHhiSSCmid monocyte precursor cells did not differ based on eGFR status. Conclusions Low eGFR was predictive for the depletion of ALDHhiSSClow primitive progenitor cells that co-express the stem cell markers CD133 and CD34 in individuals with ASCVD and/or T2D suggesting that diminished kidney function is associated with a decrease in vessel reparative pro-angiogenic progenitor cell content. Strategies that improve circulating VR cell content in individuals living with kidney and cardiometabolic disease may provide clinically meaningful benefits.Figure 1Table 1

Vascular repair and regeneration in cardiometabolic diseases.

Chronic cardiometabolic assaults during type 2 diabetes (T2D) and obesity induce a progenitor cell imbalance in the circulation characterized by overproduction and release of pro-inflammatory monocytes and granulocytes from the bone marrow alongside aberrant differentiation and mobilization of pro-vascular progenitor cells that generate downstream progeny for the coordination of blood vessel repair. This imbalance can be detected in the peripheral blood of individuals with established T2D and severe obesity using multiparametric flow cytometry analyses to discern pro-inflammatory vs. pro-angiogenic progenitor cell subsets identified by high aldehyde dehydrogenase activity, a conserved progenitor cell protective function, combined with lineage-restricted cell surface marker analyses. Recent evidence suggests that progenitor cell imbalance can be reversed by treatment with pharmacological agents or surgical interventions that reduce hyperglycaemia or excess adiposity. In this state-of-the-art review, we present current strategies to assess the progression of pro-vascular regenerative cell depletion in peripheral blood samples of individuals with T2D and obesity and we summarize novel clinical data that intervention using sodium-glucose co-transporter 2 inhibition or gastric bypass surgery can efficiently restore cell-mediated vascular repair mechanisms associated with profound cardiovascular benefits in recent outcome trials. Collectively, this thesis generates a compelling argument for early intervention using current pharmacological agents to prevent or restore imbalanced circulating progenitor content and maintain vascular regenerative cell trafficking to sites of ischaemic damage. This conceptual advancement may lead to the design of novel therapeutic approaches to prevent or reverse the devastating cardiovascular comorbidities currently associated with T2D and obesity.

P317A novel role of SGLT2 inhibitors to increase circulating proangiogenic progenitor cells in patients with type 2 diabetes and cardiovascular disease: A sub-study of the EMPA-HEART CardioLink-6 Trial

Abstract Background SGLT2 inhibitors (SGLT2i) have been demonstrated to reduce major adverse cardiovascular events and mortality in patients with type 2 diabetes (T2D) who are at high risk for cardiovascular disease (CVD). However, the mechanism(s) of the underlying benefit remain unclear. Since regenerative cell exhaustion resulting in impaired vascular homeostasis has been proposed as a key driver of CV events in T2D, we hypothesised that modulation of circulating vascular regenerative cell content by SGLT2i may be a novel basis of cardioprotection. Purpose To evaluate the effects of the SGLT2i, empagliflozin (EMPA), vs placebo on circulating vascular regenerative and pro-inflammatory cells in patients with T2D and CVD. Methods This was a biomarker sub-study of the EMPA-HEART Cardiolink-6 randomised trial of EMPA (10mg QD) vs placebo in patients with T2D and a history of coronary artery disease (prior myocardial infarction and/or coronary revascularisation). Blood samples (baseline N=48; study end N=26) underwent multiparametric progenitor cell analyses by flow cytometry. Circulating cells were assessed for aldehyde dehydrogenase (ALDH) activity, a self-protective enzyme highly expressed in several proangiogenic progenitor cell lineages, as well as cell surface co-expression of the primitive progenitor (CD34, CD133) or M1/M2 macrophage (CD80, CD163) markers. Results Individuals with increased inflammatory burden (ALDHhi granulocytes above the baseline median) were older (61±2 vs 67±2 years), more likely to be current or past smokers (21% vs 42%) and had reduced LV function, assessed by echocardiography. The placebo- and EMPA-assigned groups were equivalent at baseline with respect to the frequency and distribution of proangiogenic progenitor cells (ALDHhiSSClo), monocyte/macrophage (ALDHhiSSCmid) and inflammatory granulocyte (ALDHhiSSChi) precursors. Following 6-months of treatment with EMPA, there was a marked increase in the number of circulating primitive ALDHhiSSClo cells with CD133 (Placebo: −2.8±3.8%, EMPA: +8.6±2.5%, P<0.02) or CD133/CD34 (Placebo: 0.4±4.5%, EMPA: +13.3±3.8%, P<0.05) co-expression. Furthermore, EMPA treatment was associated with an increase in the frequency of circulating anti-inflammatory cells with M2 macrophage polarisation marked by CD163 (Placebo: −0.7±0.8%, EMPA = +3.9±1.3%, P<0.01) expression. Non-significant increases in circulating proangiogenic monocytes, and decreases in the frequency of circulating inflammatory granulocytes were also observed after EMPA treatment (vs placebo). Conclusion We provide the first evidence showing that SGLT2i treatment with EMPA alters the balance of key circulating vascular progenitor and inflammatory cells in patients with T2D and CVD. We suggest that SGLT2i may afford cardioprotection through a novel and previously unrecognised capacity to limit regenerative cell exhaustion in T2D. Acknowledgement/Funding This trial was supported by an unrestricted investigator-initiated study grant from Boehringer Ingelheim.

Association of growth-differentiation factor-15 with the number of circulating proangiogenic endothelial progenitor cells in patients with type 2 diabetes mellitus

The objective: to investigate the relationship between levels of growth differentiation factor-15 (GDF-15) and circulating number of endothelial progenitor cells (EPCs) with angiopoietin phenotypes: CD34+CD14+CD309+, and CD34+CD14+CD309+Tie2+ in patients with type 2 DM.
 Methods: The study was retrospectively involved 76 patients with type 2 DM aged 38 to 55 years and 30 healthy volunteers. Data collection included demographic and anthropometric information, hemodynamic performances and biomarkers of the diseases. Flow cytometry was used to determine EPCs' populations.
 Results: The levels of GDF-15 in peripheral blood of diabetics associated with age (r = 0.31, P = 0.044), high-sensitive C-reactive protein [hs-CRP] (r = 0.40, P = 0.001), smoking (r = 0.38, P = 0.001), body mass index [BMI] (r = 0.34, P = 0.001), LDL cholesterol (r = 0.28, P = 0.001), glycated hemoglobin [HbA1c] (r = -0.28, P = 0.001), number of CV risk factors (r = 0.26, P = 0.001). In univariate logistic regression analysis we found that level of GDF-15 ≥ 618 pg/mL, hs-CRP ≥7.12 mg/L, HbA1c ≥6.4%, fasting glucose ≥6.7 mmol/L, and BMI ≥27.3 kg/m2 predicted deficiency of both angiopoetic phenotypes of EPCs. In multivariate logistic regression model GDF-15 ≥618 pg/mL demonstrated the best odds ratio values for declining of EPCs in diabetics in comparison with other predictors including BMI, HbA1c and hs-CRP.
 Conclusion: GDF-15 was remarkably evaluated in type 2 DM population to healthy volunteers, and it was an independent factor that contributes to mobilization and probably proliferation of endothelial precursors with high angiopoetic activity.

The relationship between serum uric acid level and concentration of proangiogenic endothelial progenitor cells in chronic heart failure patients

Serum uric acid (SUA) is considered as a marker of nature progression of chronic heart failure (CHF) mediated cardiovascular remodeling. Progression of CHF associates with declining of circulating endothelial progenitor cells (EPCs) in the peripheral circulation. t he objective of this study: to establish predictive relationship between the content of uric acid in the blood and the level of circulating endothelial progenitor cells in patients with CHF of ischemic origin. Methods: The study population was structured retrospectively in 126 subjects (54 male), aged 48 to 62 years, with mild-to-severe ischemic CHFSUA level was measured by enzymatic methods, NT-pro-BNP level was examined by immunoelectrochemiluminesence method. EPCs were determined as CD 34 + cells by the flowcytometric technique using High-Definition Fluorescence Activated Cell Sorter methodology. All biomarkers were measured at baseline. The study was approved by an institutional review committee. Results: Concentrations of SUA were distributed by quartiles (Me; IQR): QI=201,1 (190,6; 223,3) umol/l; QII=275,3 (232,0; 311,0) umol/l; QIII=358,0 (320,0; 390,0) mmol/l; and QIV=449,0 (400,0; 496,0) umol/l. We found an independent impact of SUA on counts of CD14 + CD309 + EPCs (r=-0.388; P=0.001) and CD14 + CD309 + Tie2 + MPCs (r=-0.414; P=0.001), but on CD45 + CD34 + EPCs (r=-0.214; P=0.22) and CD45 - CD34 + MPCs (r=-0.16; P=0.16) did not. Cox proportional adjusted Odds Ratios analyses for CD14 + CD309 + and CD14 + CD309 + Tie2 + EPCs by SUA Quartiles (Q) has showed that high Q (Q3 and Q4) of SUA versus low Q (Q1 and Q2) associated with increased risk of depletion of both CD14 + CD309 + and CD14 + CD309 + Tie2 + EPCs. The ROC analysis showed that there was the cut-off point for the SUA level with the best prognostic potential on the risk of decreasing EPCs in both models equal 315,0 umol/l. Conclusion: Circulating level of proangiogenic EPCs phenotyped as CD14 + CD309 + and CD14 + CD309 + Tie2 + is declined progressively depended on quartiles of SUA level in CHF subjects.

Is chronic kidney disease-mineral and bone disorder associated with the presence of endothelial progenitor cells with a calcifying phenotype?

Background: Chronic kidney disease-mineral and bone disorder (CKD-MBD) has been implicated in vascular calcification pathogenesis. CKD-MBD results in alterations in the number and function of circulating endothelial progenitor cells (EPCs), physiological regulators of angiogenesis and vessel repair, commonly defined as proangiogenic progenitor cells (PACs) by the antigen pattern CD34+CD133+KDR+CD45– and putative EPCs by the pattern CD34+CD133−KDR+CD45–. These cells might acquire a calcifying phenotype in CKD-MBD, expressing mineralization biomarkers. We investigated the expression of vitamin D receptor (VDR) and osteocalcin (OC) on EPCs of healthy individuals and haemodialysis patients, and their possible associations with circulating biomarkers of inflammation and vascular calcification. Methods: We compared EPC counts, expressing VDR or OC, in 23 healthy subjects versus 53 haemodialysis patients, 17 of them without vitamin D receptor agonist (VDRA) therapy and 35 treated with calcitriol (n = 17) or paricalcitol (n = 18). The correlations with serum levels of inflammatory and calcification indexes were also analysed. Results: All subsets expressing VDR or OC were significantly higher in haemodialysis patients compared with healthy controls, but PACs were increased only in VDRA treatment subgroup, while putative EPCs showed a similar rise also in untreated patients. In VDRA-untreated patients, OC+ PACs correlated positively with calcium levels, while in VDRA-treated patients, VDR+ PACs correlated positively with interleukin 6 levels, and OC+ PACs correlated positively 25-hydroxyvitamin D levels. Conclusions: Our data suggest that in CKD-MBD, EPCs undergo an endothelial-to-procalcific shift, representing a risk factor for vascular calcification. A link between mineral disorders and vitamin D replacement therapy emerged, with potential adverse effects for CKD patients.

Human vascular progenitor cells derived from renal arteries are endothelial-like and assist in the repair of injured renal capillary networks

Vascular progenitor cells show promise for the treatment of microvasculature endothelial injury. We investigated the function of renal artery progenitor cells derived from radical nephrectomy patients, in animal models of acute ischemic and hyperperfusion injuries. Present in human adventitia, CD34positive/CD105negative cells were clonal and expressed transcription factors Sox2/Oct4 as well as surface markers CXCR4 (CD184)/KDR(CD309) consistent with endothelial progenitor cells. Termed renal artery-derived vascular progenitor cells (RAPC), injected cells were associated with decreased serum creatinine after ischemia/reperfusion, reduced albuminuria after hyperperfusion, and improved blood flow in both models. A small population of RAPC integrated with the renal microvasculature following either experimental injury. At a cellular level, RAPC promoted local endothelial migration in co-culture. Profiling of RAPC microRNA identified high levels of miRNA 218; also found at high levels in exosomes isolated from RAPC conditioned media after cell contact for 24 hours. After hydrogen peroxide-induced endothelial injury, RAPC exosomes harbored Robo-1 transcript; a gene known to be regulated by mir218. Such exosomes enhanced endothelial cell migration in culture in the absence of RAPC. Thus, our work shows the feasibility of pre-emptive pro-angiogenic progenitor cell procurement from a targeted patient population and potential therapeutic use in the form of autologous cell transplantation.

Eotaxin-Rich Proangiogenic Hematopoietic Progenitor Cells and CCR3+ Endothelium in the Atopic Asthmatic Response.

Angiogenesis is closely linked to and precedes eosinophilic infiltration in asthma. Eosinophils are recruited into the airway by chemoattractant eotaxins, which are expressed by endothelial cells, smooth muscles cells, epithelial cells, and hematopoietic cells. We hypothesized that bone marrow-derived proangiogenic progenitor cells that contain eotaxins contribute to the initiation of angiogenesis and inflammation in asthma. Whole-lung allergen challenge of atopic asthma patients revealed vascular activation occurs within hours of challenge and before airway inflammation. The eotaxin receptor CCR3 was expressed at high levels on submucosal endothelial cells in patients and a murine model of asthma. Ex vivo exposure of murine endothelial cells to eotaxins induced migration and angiogenesis. In mechanistic studies, wild-type mice transplanted with eotaxin-1/2-deficient bone marrow had markedly less angiogenesis and inflammation in an atopic asthma model, whereas adoptive transfer of proangiogenic progenitor cells from wild-type mice in an atopic asthma model into the eotaxin-1/2-deficient mice led to angiogenesis and airway inflammation. The findings indicate that Th2-promoting hematopoietic progenitor cells are rapidly recruited to the lung upon allergen exposure and release eotaxins that coordinately activate endothelial cells, angiogenesis, and airway inflammation.

Loss of endothelial-ARNT in adult mice contributes to dampened circulating proangiogenic cells and delayed wound healing.

The recruitment and homing of circulating bone marrow-derived cells include endothelial progenitor cells (EPCs) that are critical to neovascularization and tissue regeneration of various vascular pathologies. We report here that conditional inactivation of hypoxia-inducible factor's (HIF) transcriptional activity in the endothelium of adult mice (Arnt(ΔiEC) mice) results in a disturbance of infiltrating cells, a hallmark of neoangiogenesis, during the early phases of wound healing. Cutaneous biopsy punches show distinct migration of CD31(+) cells into wounds of control mice by 36 hours. However, a significant decline in numbers of infiltrating cells with immature vascular markers, as well as decreased transcript levels of genes associated with their expression and recruitment, were identified in wounds of Arnt(ΔiEC) mice. Matrigel plug assays further confirmed neoangiogenic deficiencies alongside a reduction in numbers of proangiogenic progenitor cells from bone marrow and peripheral blood samples of recombinant vascular endothelial growth factor-treated Arnt(ΔiEC) mice. In addition to HIF's autocrine requirements in endothelial cells, our data implicate that extrinsic microenvironmental cues provided by endothelial HIF are pivotal for early migration of proangiogenic cells, including those involved in wound healing.

Migratory activity of circulating progenitor cells and serum SDF-1α predict adverse events in patients with myocardial infarction

Following acute myocardial infarction (AMI), pro-angiogenic progenitor cells (PCs) are released from the bone marrow into the circulation and home to the ischaemic site attracted by a chemokine gradient. It is unknown if components of this early homeostatic response might help forecast the long-term clinical outcome. This study investigates if the number and migratory activity of circulating PCs predict adverse events in patients with AMI (clinical trial: NCT01271309). Basal counts and in vitro migratory activity of CD34/CD45/CD133/CXCR4 PCs and serum cytokine levels were assessed during the first 5 days after AMI in a consecutive series of 172 patients. Clinical outcomes of the study were death, repeat AMI, and new-onset heart failure at a 1-year follow-up. The association between PC counts and cytokine levels with the incidence of clinical outcomes was assessed by multivariable regression models. AMI patients who underwent an event showed higher serum stromal cell-derived factor 1α (SDF-1α) levels and reduced spontaneous motility of PCs in an in vitro migration assay when compared with event-free subjects. After adjustment for age, gender, the presence or absence of ST elevation, or diabetes, the percentage of PCs non-migrated towards vehicle or SDF-1α were both independent predictors of death or repeat AMI and new-onset heart failure (odds ratio [OR] = 2, P = 0.015 and OR = 1.90, P = 0.018, respectively). Moreover, serum SDF-1α levels predict adverse events (OR = 3.8, P = 0.007). Biomarkers reflecting the migratory activity of circulating PCs may aid the assessment of secondary risk in AMI patients.

Transcription factor Krüppel-like factor 2 plays a vital role in endothelial colony forming cells differentiation.

Endothelial colony forming cells (ECFCs) participate in post-natal vasculogenesis. We previously reported that vascular endothelial growth factor (VEGF) promotes human ECFC differentiation through AMP-activated protein kinase (AMPK) activation. However, the mechanisms underlying transcriptional regulation of ECFC differentiation still remain largely elusive. Here, we investigated the role of transcription factor Krüppel-like factor 2 (KLF2) in the regulation of ECFC function. Human ECFCs were isolated from cord blood and cultured. Treatment with VEGF significantly increased endothelial markers in ECFCs and their capacity for migration and tube formation. The mRNA and protein levels of KLF2 were also significantly up-regulated. This up-regulation was abrogated by AMPK inhibition or by knockdown of KLF2 with siRNA. Furthermore, adenovirus-mediated overexpression of KLF2 promoted ECFC differentiation by enhancing expression of endothelial cell markers, reducing expression of progenitor cell markers, and increasing the capacity for tube formation in vitro, indicating the important role of KLF2 in ECFC-mediated angiogenesis. Histone deacetylase 5 (HDAC5) was phosphorylated by AMPK activity induced by VEGF and the AMPK agonist AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide). In vivo angiogenesis assay revealed that overexpression of KLF2 in bone-marrow-derived pro-angiogenic progenitor cells promoted vessel formation when the cells were implanted in C57BL/6 mice. Up-regulation of KLF2 by AMPK activation constitutes a novel mechanism of ECFC differentiation, and may have therapeutic value in the treatment of ischaemic heart disease.

Nascent Endothelium Initiates Th2 Polarization of Asthma

Asthma airway remodeling is linked to Th2 inflammation. Angiogenesis is a consistent feature of airway remodeling, but its contribution to pathophysiology remains unclear. We hypothesized that nascent endothelial cells in newly forming vessels are sufficient to initiate Th2-inflammation. Vascular endothelial (VE)-cadherin is a constitutively expressed endothelial cell adhesion molecule that is exposed in its monomer form on endothelial tip cells prior to adherens junction formation. Abs targeted to VE-cadherin monomers inhibit angiogenesis by blocking this adherens junction formation. In this study, VE-cadherin monomer Ab reduced angiogenesis in the lungs of the allergen-induced murine asthma model. Strikingly, Th2 responses including, IgE production, eosinophil infiltration of the airway, subepithelial fibrosis, mucus metaplasia, and airway-hyperreactivity were also attenuated by VE-cadherin blockade, via mechanisms that blunted endothelial IL-25 and proangiogenic progenitor cell thymic stromal lymphopoietin production. The results identify angiogenic responses in the origins of atopic inflammation.

Circulating Proangiogenic Progenitor Cells Independently Predict Functional Capacity in Heart Failure Patients

BackgroundEndothelial dysfunction is an important underlying mechanism in the pathophysiology of heart failure (HF). Circulating proangiogenic progenitor cells (CPCs) are endothelial and hematopoietic progenitor cells involved in the process of vasculogenesis repairing damaged and dysfunctional endothelium. Our aim was to evaluate whether an independent association exists between CPCs and functional capacity in HF patients. MethodsThis cross-sectional study included 121 ambulatory HF patients with reduced left ventricular ejection fraction seen at a single institution. We analyzed the association between CPCs, measured as circulating CD34+VEGFR2+ cells and early outgrowth colony forming units (EO-CFUs), and patients’ functional capacity measured as peak oxygen consumption (V˙o2). ResultsThe mean age was 55 ± 11 years; 96 patients (79%) were male. Forty-three patients (36%) had ischemic cardiomyopathy. Patients were taking optimal HF therapy (96% taking β-blockers, 91% taking renin-angiotensin inhibitors, and 60% had an implanted internal cardiac defibrillator). In univariate analyses, CD34+VEGFR2+ cells were inversely associated with peak V˙o2 (P = 0.02) while EO-CFUs showed a positive association with peak V˙o2 (P < 0.01). These associations persisted after adjusting for sex, New York Heart Association class, body mass index, diabetes, cardiac resynchronization therapy, ischemic cardiomyopathy and b-type natriuretic peptide levels. ConclusionsCultured EO-CFUs may represent a measure of functional capacity and vasculogenesis potential while CD34+VEGFR2+ cells represent the mobilized cells in response to endothelial damage. Our study suggests that lower EO-CFUs (worse cell function) and higher CD34+VEGFR2+ cells are associated with poorer functional capacity.

Dysregulated Arginine Methylation Is Associated with Deregulated Proangiogenic Progenitor Activity and Reduced Adaptive Vascular Response in Patients with Advanced Left Sided Heart Failure

Asymmetric Di-methyl Arginine (ADMA) is a marker of endothelial dysfunction and is also an independent prognostic indicator of morbidity in Heart Failure (HF). Proangiogenic Progenitor Cells (PPC's) have been detected in the vasculature of pulmonary arterial hypertension (PAH) patients. These PPC's demonstrated hyper proliferative and deregulated growth. Whether such deregulated angiogenic activity of PPC's occurs in patients with left sided HF and elevated mean pulmonary artery pressures (mPAP) and how this correlates with dysregulated arginine methylation remains unclear.

Heme oxygenase-1 is crucial for proangiogenic progenitor cells and facilitates cell-free strategy for therapeutic angiogenesis of ischemic limbs in diabetes

Heme oxygenase-1 is crucial for proangiogenic progenitor cells and facilitates cell-free strategy for therapeutic angiogenesis of ischemic limbs in diabetes

Exercise training in intermittent claudication: Effects on antioxidant genes, inflammatory mediators and proangiogenic progenitor cells

Exercise training remains a therapy of choice in intermittent claudication (IC). However, too exhaustive exercise may cause ischaemic injury and inflammatory response. We tested the impact of three-month treadmill training and single treadmill exercise on antioxidant gene expressions, cytokine concentrations and number of marrow-derived proangiogenic progenitor cells (PPC) in the blood of IC patients. Blood samples of 12 patients were collected before and after training, before and 1, 3 and 6 hours after the single exercise. PPCs were analysed with flow cytometry, cytokine concentrations were checked with Milliplex MAP, while expression of mRNAs and miRNAs was evaluated with qRT-PCR. Treadmill training improved pain-free walking time (from 144 ± 44 seconds [s] to 311 ± 134 s, p=0.02) and maximum walking time (from 578 ± 293 s to 859 ± 423 s, p=0.01) in IC patients. Before, but not after training, the single treadmill exercise increased the number of circulating CD45dimCD34+CD133-KDR+ PPCs (p=0.048), decreased expression of HMOX1 (p=0.04) in circulating leukocytes, reduced tumour necrosis factor-α (p=0.03) and tended to elevate myeloperoxidase (p=0.06) concentrations in plasma. In contrast, total plasminogen activator inhibitor-1 was decreased by single exercise only after, but not before training (p=0.02). Both before and after training the single exercise decreased monocyte chemoattractant protein (MCP)-1 (p=0.006 and p=0.03) concentration and increased SOD1 (p=0.001 and p=0.01) expression. Patients after training had also less interleukin-6 (p=0.03), but more MCP-1 (p=0.04) in the blood. In conclusion, treadmill training improves walking performance of IC patients, attenuates the single exercise-induced changes in gene expressions or PPC mobilisation, but may also lead to higher production of some proinflammatory cytokines.

The Role of Chemokines, Cytokines and Adhesion Molecules in Stem Cell Trafficking and Homing

Although definition and mechanistic understanding of pro-angiogenic progenitor cells remains unsatisfactory, general agreement highlights their role in regenerative process following tissue injury and ischemia. Furthermore, stem-cell based therapy represents a hot topic of cardiovascular medicine. Recent studies provide new insights on the signalling pathways that modulate stem/progenitor cell mobilization from bone marrow, homing to ischemic area and participation in vascular remodelling and tissue healing. This review focuses on current knowledge and emerging concepts on stem cell/progenitor cell trafficking in relation to changes in surrounding environment and epigenetic modifications caused by risk factors and comorbidities.

Pro-angiogenic hematopoietic progenitor cells and endothelial colony-forming cells in pathological angiogenesis of bronchial and pulmonary circulation

Dysregulation of angiogenesis is a common feature of many disease processes. Vascular remodeling is believed to depend on the participation of endothelial progenitor cells, but the identification of endothelial progenitors in postnatal neovascularization remains elusive. Current understanding posits a role for circulating pro-angiogenic hematopoietic cells that interact with local endothelial cells to establish an environment that favors angiogenesis in physiologic and pathophysiologic responses. In the lung, increased and dysregulated angiogenesis is a hallmark of diseases of the bronchial and pulmonary circulations, manifested by asthma and pulmonary arterial hypertension (PAH), respectively. In asthma, T(Helper)-2 immune cells produce angiogenic factors that mobilize and recruit pro-inflammatory and pro-angiogenic precursors from the bone marrow into the airway wall where they induce angiogenesis and fuel inflammation. In contrast, in PAH, upregulation of hypoxia-inducible factor (HIF) in vascular cells leads to the production of bone marrow-mobilizing factors that recruit pro-angiogenic progenitor cells to the pulmonary circulation where they contribute to angiogenic remodeling of the vessel wall. This review focuses on current knowledge of pro-angiogenic progenitor cells in the pathogenesis of asthma and PAH.

Abstract 3429: Promotion of metastasis by frequently used chemotherapeutic agents

Abstract It is commonly assumed that early cancer progression during chemotherapy is part of the natural course of disease. However, there is increasing evidence that chemotherapy, in addition to its cytotoxic effects, may also confer tumor-favorable signals as part of a host response. Prime examples are increases in circulating, pro-angiogenic endothelial progenitor cells (EPCs) and growth factors after chemotherapy. We hypothesize that when tumors can overcome the cytotoxic effects of chemotherapy through resistance, these tumor-favorable signals will obfuscate the benefits of treatment and may actually facilitate metastatic spread. In order to study therapy-induced metastasis formation in absence of direct cytotoxic effects on tumor cells, mice were pre-treated with chemotherapy. Four days later, when the cytotoxic agents had been cleared from circulation, tumor cells were injected intravenously. Thirteen days after tumor cell injection, lung metastases had formed in untreated mice (5.4±3.9 surface metastases). Remarkably, pre-treatment with two chemotherapeutic agents that are frequently used in clinical practice, paclitaxel and cisplatin, showed a robust increase in the number of lung colonies compared to untreated mice (12.7±6.9, p=0.0003 for paclitaxel and 32.7±9.9, p&amp;lt;0.0001 for cisplatin). Similar effects were observed when using a different tumor cell line in a second mouse strain. Additional experiments in immune deficient Rag2−/−γc−/− mice showed that suppression of the immune system by chemotherapy does not account for this effect, as chemotherapy still enhanced metastasis formation in these mice (5.5±4.0 surface metastases in untreated mice compared to 28.3±11.8 in cisplatin pre-treated mice, p=0.01). Nevertheless, combining chemotherapy with antibodies against the murine vascular endothelial growth factor receptor 2 (VEGFR2) fully abrogated the chemotherapy-induced increase in metastases (3.0±3.7, p=0.0004 compared to chemotherapy alone). This implies that chemotherapy has the ability to signal through the VEGFR2 pathway to promote metastasis. Whether this is due to local pro-angiogenic effects in the lungs, or to a systemic mobilization of VEGFR2-expressing EPCs into the circulation, is currently under investigation. In conclusion, we show that two widely used chemotherapeutic drugs, with different mechanisms of action, are able to increase metastasis formation when direct cytotoxic effects on tumor cells are absent. Targeting VEGFR2 interferes with the enhanced metastatic pattern. These data provide novel evidence for interactions between chemotherapy and the microenvironment, which could potentially lead to early progression in patients with chemoresistant tumors. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3429.

Augmented Revascularization and Recovery From Acute Limb Ischemia After Transplantation of Human Umbilical Cord Blood Progenitor Cells with High Aldehyde Dehydrogenase Activity.

Abstract 3042Poster Board II-1018Clinical application of regenerative cell-based therapies for the treatment of ischemic vascular disease has proven challenging to implement due the involvement of multiple human cell types that co-ordinate angiogenesis. Previously, transplanted human progenitor cells from hematopoietic, endothelial, and mesenchymal lineages have all been implicated in the recovery and de novo production of perfused blood vessels in vivo. Using high aldehyde dehydrogenase (ALDHhi) activity, a conserved function of these pro-angiogenic hematopoietic and non-hematopoietic progenitor cell lineages, we have recently shown that transplanted human bone marrow (BM) ALDHhi cells transiently recruit to areas of hypoxia and augment revascularization and recovery of perfusion in ischemic limbs via the stimulation of endogenous vascular regeneration. However, clinical use of autologous BM-derived cells in patients with critical limb ischemia may be limited by the availability or dysfunction of transplanted pro-angiogenic cells due to vascular disease-related pathologies. The non-invasive collection and early ontogeny of human umbilical cord blood (UCB) progenitor cells provides a promising alternative source of pro-angiogenic progenitor cells with vascular regenerative functions. Human UCB mononuclear cells (MNC) were purified using high-speed fluorescence-activated cell sorting to accrue clinically applicable cell populations with low side scatter and high (ALDHhi, 0.4±0.1% of total MNC) versus low (ALDHlo, 41.2±3.6% of total MNC) ALDH activity (n=10). Compared to UCB-derived ALDHlo cells, which were devoid of hematopoietic and non-hematopoietic progenitor function in vitro, ALDHhi cells were enriched for hematopoietic colony forming cells (1 HCFC in 3.6 cells, n=4) and endothelial colony forming cells (1 ECFC in 5.8×104 cells, n=4) enumerated after 14 days in culture. In contrast to human BM-derived ALDH-purified cells which were enriched for mesenchymal colony forming cells, analogous ALDHlo and ALDHhi cells from UCB did not establish mesenchymal-stromal colonies in vitro suggesting a comparative deficiency in mesenchymal stem cell frequency after UCB venipuncture. We investigated the vascular regenerative capacity of these human UCB-derived ALDHlo and ALDHhi cells by tail-vein transplantation into sublethally irradiated (275cGy) immune-deficient, β-glucuronidase (GUSB) deficient, NOD/SCID/MPSVII mice with acute limb ischemia induced by unilateral femoral artery ligation and transection. Using weekly laser Doppler perfusion imaging to track the kinetics of blood flow recovery indicated by the perfusion ratio (PR) in the ischemic versus non-ischemic limb, mice transplanted with 2×105-4×105 purified ALDHhi cells showed significantly improved recovery of limb perfusion by day 21 post-transplantation (PR=0.70±0.06, n=6) compared to mice injected with phosphate-buffered saline (PR=0.35±0.06, n=5, P<0.01), 10×106 ALDHlo cells (PR=0.45±0.07, n=7, P<0.01), or 20×106 unpurified UCB MNC (PR=0.40±0.06, n=7, P<0.01). Furthermore, transplantation of ALDHhi progenitor cells augmented revascularization in the ischemic limb compared to all other treatments at day 28 post-transplantation (P<0.01), quantified by immunohistochemical detection and enumeration of von Willebrand factor+ blood vessels in frozen sections from the ischemic adductor muscle. Only mice transplanted with ALDHhi cells showed retention of human hematopoietic (CD45+/HLA-A,B,C+) cells in the ischemic adductor muscle for 28 days (3.0±2.1%, n=6) quantified by flow cytometry. Ischemic muscle sections from NOD/SCID/MPSVII mice transplanted with human UCB ALDHhi cells confirmed human cell retention and engraftment adjacent to damaged vasculature and ischemic muscle fibres at day 28 post-transplantation, measured by colorimetric detection of ubiquitous GUSB activity in transplanted human cells. Human UCB-derived ALDHhi cells, which were devoid of mesenchymal-stromal cells but enriched for potentially pro-angiogenic endothelial and hematopoietic progenitor lineages, showed persistent retention in areas of hypoxia and augmented the endogenous revascularization and recovery of perfusion in ischemic limbs. Thus, ALDHhi mixed progenitor cells may prove to be a useful allogeneic alternative to human BM in the clinical treatment of patients with severe peripheral vascular disease. DisclosuresNo relevant conflicts of interest to declare.