Circulating Angiogenic Cells Research Articles

Enhanced endoplasmic reticulum stress in bone marrow angiogenic progenitor cells in a mouse model of long-term experimental type 2 diabetes.

Bone marrow-derived circulating angiogenic cells (CACs) play an important role in vascular repair. In diabetes, compromised functioning of the CACs contributes to the development of diabetic retinopathy; however, the underlying mechanisms are poorly understood. We examined whether endoplasmic reticulum (ER) stress, which has recently been linked to endothelial injury, is involved in diabetic angiogenic dysfunction. Flow cytometric analysis was used to quantify bone marrow-derived progenitors (Lin(-)/c-Kit(+)/Sca-1(+)/CD34(+)) and blood-derived CACs (Sca-1(+)/CD34(+)) in 15-month-old Lepr (db) (db/db) mice and in their littermate control (db/+) mice used as a model of type 2 diabetes. Markers of ER stress in diabetic (db/db) and non-diabetic (db/+) bone marrow-derived early outgrowth cells (EOCs) and retinal vascular density were measured. The numbers of bone-marrow progenitors and CACs were significantly reduced in db/db mice. Vascular density was markedly decreased in the retinas of db/db mice, and this was accompanied by vascular beading. Microglial activation was enhanced, as was the production of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). The production of ER stress markers (glucose-regulated protein-78 [GRP-78], phosphorylated inositol-requiring enzyme-1α [p-IRE-1α], phosphorylated eukaryotic translation initiation factor-2α [p-eIF2α], activating transcription factor-4 [ATF4], C/EBP homologous protein [CHOP] and spliced X-box binding protein-1 [XBP1s]) was significantly increased in bone marrow-derived EOCs from db/db mice. In addition, mouse EOCs cultured in high-glucose conditions demonstrated higher levels of ER stress, reduced colony formation, impaired migration and increased apoptosis, all of which were largely prevented by the chemical chaperone 4-phenylbutyrate. Taken together, our results indicate that diabetes increases ER stress in bone marrow angiogenic progenitor cells. Thus, targeting ER stress may offer a new approach to improving angiogenic progenitor cell function and promoting vascular repair in diabetes.

Chronic endurance exercise affects paracrine action of CD31+ and CD34+ cells on endothelial tube formation.

We aimed to determine if chronic endurance-exercise habits affected redox status and paracrine function of CD34(+) and CD34(-)/CD31(+) circulating angiogenic cells (CACs). Subjects were healthy, nonsmoking men and women aged 18-35 yr and categorized by chronic physical activity habits. Blood was drawn from each subject for isolation and culture of CD34(+) and CD34(-)/CD31(+) CACs. No differences in redox status were found in any group across either cell type. Conditioned media (CM) was generated from the cultured CACs and used in an in vitro human umbilical vein endothelial cell-based tube assay. CM from CD34(+) cells from inactive individuals resulted in tube structures that were 29% shorter in length (P < 0.05) and 45% less complex (P < 0.05) than the endurance-trained group. CD34(-)/CD31(+) CM from inactive subjects resulted in tube structures that were 26% shorter in length (P < 0.05) and 42% less complex (P < 0.05) than endurance-trained individuals. Proteomics analyses identified S100A8 and S100A9 in the CM. S100A9 levels were 103% higher (P < 0.05) and S100A8 was 97% higher in the CD34(-)/CD31(+) CM of inactive subjects compared with their endurance-trained counterparts with no significant differences in either protein in the CM of CD34(+) CACs as a function of training status. Recombinant S100A8/A9 treatment at concentrations detected in inactive subjects' CD34(-)/CD31(+) CAC CM also reduced tube formation (P < 0.05). These findings are the first, to our knowledge, to demonstrate a differential paracrine role in CD34(+) and CD34(-)/CD31(+) CACs on tube formation as a function of chronic physical activity habits and identifies a differential secretion of S100A9 by CD34(-)/CD31(+) CACs due to habitual exercise.

Changes in endothelial progenitor cell subsets in normal pregnancy compared with preeclampsia

BackgroundThe results of studies measuring the number of endothelial progenitor cells (EPCs) in normal pregnancies and in preeclampsia have been highly controversial or even contradictory because of cross-sectional designs and different methodologies enumerating three distinct subsets of EPCs: circulating angiogenic cells (CAC), colony-forming unit endothelial cell (CFU-ECs), and endothelial colony forming cells (ECFCs). To provide a clear explanation for these underlying controversies, we designed a prospective study to compare the number of all EPC subsets between three trimesters of normal gestation and a case–control study to compare these values as preeclampsia occurs with those from gestational age (GA) matched normal pregnancy. MethodsSamples from peripheral blood of nine women were taken during their three consecutive trimesters of normal pregnancy, and from eight women with preeclampsia. To cover most of the reported phenotypes for CACs and ECFCs in the literature, we enumerated 13 cell populations by quantitative flow cytometry using various combinations of the markers CD34, CD133, CD309, and CD45. We used routine culturing techniques to enumerate CFU-ECs. ResultsThe numbers of CACs and ECFCs were higher in women with preeclampsia (p = 0.014). By contrast, preeclampsia was associated with a reduced number of CFU-ECs (p = 0.039). The CAC number rose with the increase in GA (p = 0.016) during normal pregnancy, while the number of CFU-ECs and ECFCs did not differ during the trimesters. ConclusionAlthough we did demonstrate an increase in absolute counts of CACs and ECFCs in preeclampsia, fewer colony formation capacities indicated a loss in their functional capabilities. By contrast, the number of CACs increased without alterations in colony formation ability in normal pregnancy with the growth of the fetus. Here, by comparing different methodologies to calculate the number of EPC subsets, we could imitate the existing controversy in the literature for such calculations, which may help to elucidate clearer explanations.

Abstract 643: Impaired Integrin β3 Delays Endothelial Cell Regeneration and Contributes to Arteriovenous Graft Failure in mice

Objective: Neointima formation is associated with stenosis and subsequent thrombosis in arteriovenous grafts (AVG). A role of integrin β3 in the neointima formation of AVGs remains poorly understood. Approach and Results: In integrin β3 -/- mice, we found significantly accelerated occlusion of AVGs compared to the wild type mice. This is caused by the development of neointima and lack of endothelial regeneration. The latter is a direct consequence of impaired functions of circulating angiogenic cells (CACs) and platelets in integrin β3 -/- mice. Evidence suggests the involvement of platelet regulating CAC homing to and differentiation at graft sites via TGF-β1 and Notch signaling pathway. First, CACs deficient of integrin β3 impaired adhesion activity toward exposed subendothelium. Second, platelets from integrin β3 -/- mice failed to sufficiently stimulate CACs to differentiate into mature endothelial cells. Finally, we found that TGF-β1 level was increased in platelets from integrin β3 -/- mice and resulted in enhanced Notch1 activation in CACs in AVGs. These results demonstrate that integrin β3 is critical for endothelial cell homing and differentiation. The increased TGF-β1 and Notch1 signaling mediates integrin β3 -/- -induced AVG occlusion. This accelerated occlusion of AVGs was reversed in integrin β3 -/- mice transplanted with the bone marrow from WT mice. Conclusion: Our results suggest that boosting integrin β3 function in the endothelial cells and platelets could prevent neointima and thrombosis in AVGs.

Abstract 236: Vascular Smooth Muscle Cells Stimulate Re-endothelialization Through Protein Kinase C-delta-dependent Release Of CXCL7 and Recruitment of Circulating Angiogenic Cells

Objective: In response of injury, vascular smooth muscle cells (VSMCs) undergo proliferation, migration, as well as apoptosis. We have previously shown that gene transfer of a pro-apoptotic mediator protein kinase C-delta (PKCδ) reduces intimal hyperplasia. Subsequently, we showed that VSMCs are a rich source of chemokines. In this study, we explored whether VSMCs may play a role in regulation of re-endothelialization through PKCδ-dependent release of chemokines. Methods and Results: We constructed an adenovirus that expresses PKCδ under VSMC-specific SM22 promoter (AdPKCδ) and use it to drive PKCδ expression in VSMCs of injured rat carotid arteries. Compared to the empty vector (AdNull), AdPKCδ accelerated re-endothelialization, reflected by a larger area that was excluded of Evan’s blue (25.38±7.52% v.s 59.60±5.01%). Media conditioned by PKCδhigh VSMCs had no measurable effects on endothelial cell functions, including BrdU incorporation, transwell migration, scratch wound healing, and tube formation. Interestingly, PKCδhigh VSMCs conditioned media attracted circulating angiogenic cells (CACs), a population of cells that promote neovascularization via production of angiogenic factors. Using a PCR array analysis, we identified a group of PKCδ upregulated chemokines in VSMCs, including MCP-1, CXCL1, and CXCL7. Neutralizing CXCL7, but not MCP-1 or CXCL1, significantly blocked PKCδhigh VSMC conditioned media induced migration of CACs. In vivo, we observed more CD133+ (a marker of CACs) cells lined up to the lumen of PKCδhigh expressing vessels, while the number of CD133+ cells was comparable in the blood of AdPKCδ treated rats and AdNull treated rats. Immunofluorescence staining revealed that delivery of PKCδ gene increased CXCL7 expression in media VSMCs. Conclusions: Our data suggest that VSMCs stimulate re-endothelialization through PKCδ-dependent release of CXCL7 and recruitment of CACs.

Effects of TiO2 and Co3O4 Nanoparticles on Circulating Angiogenic Cells

Background and AimSparse evidence suggests a possible link between exposure to airborne nanoparticles (NPs) and cardiovascular (CV) risk, perhaps through mechanisms involving oxidative stress and inflammation. We assessed the effects of TiO2 and Co3O4 NPs in human circulating angiogenic cells (CACs), which take part in vascular endothelium repair/replacement.MethodsCACs were isolated from healthy donors’ buffy coats after culturing lymphomonocytes on fibronectin-coated dishes in endothelial medium for 7 days. CACs were pre-incubated with increasing concentration of TiO2 and Co3O4 (from 1 to 100 μg/ml) to test the effects of NP – characterized by Transmission Electron Microscopy – on CAC viability, apoptosis (caspase 3/7 activation), function (fibronectin adhesion assay), oxidative stress and inflammatory cytokine gene expression.ResultsNeither oxidative stress nor cell death were associated with exposure to TiO2 NP (except at the highest concentration tested), which, however, induced a higher pro-inflammatory effect compared to Co3O4 NPs (p<0.01). Exposure to Co3O4 NPs significantly reduced cell viability (p<0.01) and increased caspase activity (p<0.01), lipid peroxidation end-products (p<0.05) and pro-inflammatory cytokine gene expression (p<0.05 or lower). Notably, CAC functional activity was impaired after exposure to both TiO2 (p<0.05 or lower) and Co3O4 (p<0.01) NPs.ConclusionsIn vitro exposure to TiO2 and Co3O4 NPs exerts detrimental effects on CAC viability and function, possibly mediated by accelerated apoptosis, increased oxidant stress (Co3O4 NPs only) and enhancement of inflammatory pathways (both TiO2 and Co3O4 NPs). Such adverse effects may be relevant for a potential role of exposure to TiO2 and Co3O4 NPs in enhancing CV risk in humans.

Impaired Endothelial Regeneration Through Human Parvovirus B19-Infected Circulating Angiogenic Cells in Patients With Cardiomyopathy.

Human parvovirus B19 (B19V) is a common pathogen in microvascular disease and cardiomyopathy, owing to infection of endothelial cells. B19V replication, however, is almost restricted to erythroid progenitor cells (ErPCs). Endothelial regeneration attributable to bone marrow-derived circulating angiogenic cells (CACs) is a prerequisite for organ function. Because of many similarities of ErPCs and CACs, we hypothesized that B19V is a perpetrator of impaired endogenous endothelial regeneration. B19V DNA and messenger RNA from endomyocardial biopsy specimens, bone marrow specimens, and circulating progenitor cells were quantified by polymerase chain reaction analysis. The highest B19V DNA concentrations were found in CD34(+)KDR(+) cells from 17 patients with chronic B19V-associated cardiomyopathy. B19V replication intermediates could be detected in nearly half of the patients. Furthermore, chronic B19V infection was associated with impaired endothelial regenerative capacity. B19V infection of CACs in vitro resulted in expression of transcripts encoding B19V proteins. The capsid protein VP1 was identified as a novel inducer of apoptosis, as were nonstructural proteins. Inhibition studies identified so-called death receptor signaling with activation of caspase-8 and caspase-10 to be responsible for apoptosis induction. B19V causally impaired endothelial regeneration with spreading of B19V in CACs in an animal model in vivo. We thus conclude that B19V infection and damage to CACs result in dysfunctional endogenous vascular repair, supporting the emergence of primary bone marrow disease with secondary end-organ damage.

Development of reporter gene imaging techniques for long-term assessment of human circulating angiogenic cells

The use of biomaterials and tracking the long-term fate of the transplanted cells is expected to help improve the clinical translation of cell therapies for cardiac regeneration. To this end, reporter gene strategies are promising for monitoring the fate of cells transplanted with or without a delivery biomaterial; however, their application with primary adult progenitor cells (such as human circulating angiogenic cells (CACs)) has not been extensively evaluated. In this study, human CACs were transduced with reporter genes via one of two lentiviral (LV) vectors: LV-GFP-iresTK or LV-Fluc-RFP-tTK. The mean transduction efficiency was 15% (LV-GFP-iresTK) and 13% (LV-Fluc-RFP-tTK) at multiplicities of infection (MOI) of 10 and 50, respectively. Western blot analysis confirmed HSV1-tk protein expression in transduced CACs. There was no significant difference in viability between the transduced CACs and the untreated controls at a MOI of 50 or below. However, a reduction was observed in cell viability of CACs transduced with LV-Fluc-RFP-tTK at an MOI of 100. Cell migration and angiogenic potential were not affected by transduction protocol. After 4 weeks, 80.3 ± 8.4% of the labeled cells continued to express the reporters and could be visualized when embedded within a collagen matrix scaffold. Therefore, quiescent human CACs can be stably transduced to express reporter genes without affecting their function. This reporter gene technique is a promising approach to be further tested for tracking transplanted CACs (±delivery matrix) non-invasively and longitudinally.

Local delivery of VEGF and SDF enhances endothelial progenitor cell recruitment and resultant recovery from ischemia.

Biomaterials may improve outcomes of endothelial progenitor-based therapies for the treatment of ischemic cardiovascular disease, due to their ability to direct cell behavior. We hypothesized that local, sustained delivery of exogenous vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF) from alginate hydrogels could increase recruitment of systemically infused endothelial progenitors to ischemic tissue, and subsequent neovascularization. VEGF and SDF were found to enhance in vitro adhesion and migration of outgrowth endothelial cells (OECs) and circulating angiogenic cells (CACs), two populations of endothelial progenitors, by twofold to sixfold, and nearly doubled recruitment to both ischemic and nonischemic muscle tissue in vivo. Local delivery of VEGF and SDF to ischemic hind-limbs in combination with systemic CAC delivery significantly improved functional perfusion recovery over OEC delivery, or either treatment alone. Compared with OECs, CACs were more responsive to VEGF and SDF treatment, promoted in vitro endothelial sprout formation in a paracrine manner more potently, and demonstrated greater influence on infiltrating inflammatory cells in vivo. These studies demonstrate that accumulation of infused endothelial progenitors can be enriched using biomaterial-based delivery of VEGF and SDF, and emphasize the therapeutic benefit of using CACs for the treatment of ischemia.

Impaired integrin β3 delays endothelial cell regeneration and contributes to arteriovenous graft failure in mice.

Neointima formation is associated with stenosis and subsequent thrombosis in arteriovenous grafts (AVGs). A role of integrin β3 in the neointima formation of AVGs remains poorly understood. In integrin β3(-/-) mice, we found significantly accelerated occlusion of AVGs compared with the wild-type mice. This is caused by the development of neointima and lack of endothelial regeneration. The latter is a direct consequence of impaired functions of circulating angiogenic cells (CACs) and platelets in integrin β3(-/-) mice. Evidence suggests the involvement of platelet regulating CAC homing to and differentiation at graft sites via transforming growth factor-β1 and Notch signaling pathway. First, CACs deficient of integrin β3 impaired adhesion activity toward exposed subendothelium. Second, platelets from integrin β3(-/-) mice failed to sufficiently stimulate CACs to differentiate into mature endothelial cells. Finally, we found that transforming growth factor-β1 level was increased in platelets from integrin β3(-/-) mice and resulted in enhanced Notch1 activation in CACs in AVGs. These results demonstrate that integrin β3 is critical for endothelial cell homing and differentiation. The increased transforming growth factor-β1 and Notch1 signaling mediates integrin β3(-/-)-induced AVG occlusion. This accelerated occlusion of AVGs was reversed in integrin β3(-/-) mice transplanted with the bone marrow from wild-type mice. Our results suggest that boosting integrin β3 function in the endothelial cells and platelets could prevent neointima and thrombosis in AVGs.

Impaired Circulating Angiogenic Cells Mobilization and Metalloproteinase-9 Activity after Dynamic Exercise in Early Metabolic Syndrome.

Increased levels of adhesion molecules or metalloproteinases (MMPs) may indicate endothelial dysfunction. Exercise mobilizes circulating angiogenic cells (CACs) from bone marrow in healthy subjects, improving vascular function. However, it is unclear whether this mechanism is preserved in the early stages of metabolic syndrome (early MetS). We aimed to evaluate the acute effects of exercise on adhesion molecules, angiogenic factors, MMPs, and CACs in early MetS. Fifteen subjects with early MetS and nine healthy controls underwent an exercise session and a nonexercise session, randomly. Adhesion molecules, angiogenic factors, CACs, and MMPs were evaluated before and after exercise or nonexercise sessions. At baseline, levels of sE-selectin, sICAM-1, and MMP-9 were higher in early MetS than in controls (P ≤ 0.03). After exercise, sE-selectin, sICAM-1, and MMP-9 levels were still higher in early MetS (P < 0.05). Subjects with early MetS presented less CACs (P = 0.02) and higher MMP-9 activity (P ≤ 0.04), while healthy controls presented higher MMP-2 activity after exercise. There was no difference between moments in nonexercise session (P > 0.05). In conclusion, subjects with early MetS already presented impaired endothelial function at rest along with a decrease in CACs and an increase in MMP-9 activity in response to exercise.

Absence of Correlation between Changes in the Number of Endothelial Progenitor Cell Subsets.

Background and ObjectivesPreviously, various methodologies were used to enumerate the endothelial progenitor cells (EPCs). We now know that these methodologies enumerate at least three different EPC subsets: circulating angiogenic cells (CACs), colony-forming unit endothelial cells (CFU-ECs), and endothelial colony-forming cells (ECFCs). It is not clear whether there is a correlation between changes in the number of these subsets. The aim of the current study is to find an answer to this question.Materials and MethodsThe number of all EPC subsets was quantified in the peripheral blood of nine pregnant women in their first and third trimesters of pregnancy. We enumerated 14 cell populations by quantitative flow-cytometry using various combinations of the markers, CD34, CD133, CD309, and CD45, to cover most of the reported phenotypes of CACs and ECFCs. Culturing technique was used to enumerate the CFU-ECs. Changes in the number of cells were calculated by subtracting the number of cells in the first trimester peripheral blood from the number of cells in the third trimester peripheral blood, and correlations between these changes were analyzed.ResultsThe number of CFU-ECs did not correlate with the number of ECFCs and CACs. Also, CACs and ECFCs showed independent behaviors. However, the number of CACs showed a strong correlation with the number of CD133+CD309+ cells (p=0.001) and a moderate correlation with the number of CD34+CD309+ cells (p=0.042). Also, the number of ECFCs was correlated with the number of CD309+CD45- cells (p=0.029) and CD34+CD45- cells (p=0.03).ConclusionOur study showed that the three commonly used methods for quantifying EPC subsets represent different cells with independent behaviors. Also, any study that measured the number of EPCs using the flow cytometry method with a marker combination that lacks CD309 may be inaccurate.

Central role of eNOS in the maintenance of endothelial homeostasis.

Disruption of endothelial function is considered a key event in the development and progression of atherosclerosis. Endothelial nitric oxide synthase (eNOS) is a central regulator of cellular function that is important to maintain endothelial homeostasis. Endothelial homeostasis encompasses acute responses such as adaption of flow to tissue's demand and more sustained responses to injury such as re-endothelialization and sprouting of endothelial cells (ECs) and attraction of circulating angiogenic cells (CAC), both of which support repair of damaged endothelium. The balance and the intensity of endothelial damage and repair might be reflected by changes in circulating endothelial microparticles (EMP) and CAC. Flow-mediated vasodilation (FMD) is a generally accepted clinical read-out of NO-dependent vasodilation, whereas EMP are upcoming prognostically validated markers of endothelial injury and CAC are reflective of the regenerative capacity with both expressing a functional eNOS. These markers can be integrated in a clinical endothelial phenotype, reflecting the net result between damage from risk factors and endogenous repair capacity with NO representing a central signaling molecule. Improvements of reproducibility and observer independence of FMD measurements and definitions of relevant EMP and CAC subpopulations warrant further research. Endothelial homeostasis may be a clinical therapeutic target for cardiovascular health maintenance.

Collagen matrix-induced expression of integrin αVβ3 in circulating angiogenic cells can be targeted by matricellular protein CCN1 to enhance their function.

Circulating angiogenic cells (CACs) play an important role in vascular homeostasis and hold therapeutic promise for treating a variety of cardiovascular diseases. However, further improvements are needed because the effects of CAC therapy remain minimal or transient. The regenerative potential of these cells can be improved by culture on a collagen-based matrix through the up-regulation of key integrin proteins. We found that human CAC function was enhanced by using the matricellular protein CCN1 (CYR61/CTGF/NOV family member 1) to target integrin αV and β3, which are up-regulated on matrix. Compared to matrix-cultured CACs, CCN1-matrix CACs exhibited a 2.2-fold increase in cell proliferation, 1.8-fold greater migration toward VEGF, and 1.7-fold more incorporation into capillary-like structures in an angiogenesis assay. In vivo, intramuscular injection of CCN1-matrix-cultured CACs into ischemic hind limbs of CD-1 nude mice resulted in blood flow recovery to 80% of baseline, which was greater than matrix-cultured CACs (66%) and PBS (35%) treatment groups. Furthermore, transplanted CCN1-matrix CACs exhibited greater engraftment (11-fold) and stimulated the up-regulation of survival and angiogenic genes (>3-fold). These findings reveal the importance of cell-matrix interactions in regulating CAC function and also reveal a mechanism by which these may be exploited to enhance cell therapies for ischemic disease.

Abstract 13347: Injectable Collagen-based Hydrogel Matrix Modulates Micro-RNA Expression and Prevents Cardiac Deterioration Post-myocardial Infarction

Background: Injectable hydrogel biomaterials have emerged as promising therapies for treating myocardial infarction (MI). We developed a collagen type I based injectable hydrogel matrix that can prevent the deterioration of cardiac function when delivered soon post-MI and found that the effects may be mediated through a microRNA (miRNA) mechanism. Methods/Results: C57BL/6J mice underwent LAD ligation to induce MI. Mice then received myocardial injections of PBS or matrix delivered at 3hours post-MI. Analyses were performed at 2 days, 1 and 3 months post-treatment. At one month post-treatment, mice that received the matrix had superior left ventricular ejection fraction (LVEF; 45.1±2.3%) compared to the PBS group (29.6±2.4%; p&lt; 0.001). LVEF was maintained in matrix-treated mice at 3 months (42.9±3.8%). Matrix treatment was also associated with reduced infarct sizes and improved ventricular volumes. Matrix-treated mice had more angiogenesis, mitigated apoptosis and reduced inflammation in the infarcted myocardium at both 2 and 28 days post-treatment. To better understand the mechanisms, we performed miRNA microarrays on infarct and peri-infarct tissue. Matrix treatment resulted in 120 miRNAs with differential expression within +/- 0.3 log2 fold change. In particular, we found matrix treatment down-regulated miR-92a ( p&lt; 0.0005), an anti-angiogenic miRNA. Integrins α5 (Itgα5) and αV (ItgαV), involved in angiogenesis and cell-matrix interactions, were identified as putative miR-92a targets and pursued further in vitro using circulating angiogenic cells (CACs). CACs cultured on the matrix had increased Itgα5 and ItgαV expression after 4 days (12.4-fold and 13.9-fold, respectively vs. fibronectin; p&lt; 0.01). When applied in an in vitro angiogenesis assay, the number of CACs that incorporated into capillary-like structures was greater (by 4.2-fold) for cells derived from matrix culture ( p &lt;0.005). Conclusion: We demonstrate pronounced benefits associated with our hydrogel matrix when delivered at 3h post-MI. The matrix effects may be mediated, at least in part, through its ability to regulate miR-92a and integrin-mechanisms. Overall, the matrix may provide a promising therapeutic approach for protecting the myocardium post-MI.

SINGLE CELL ENCAPSULATION IMPROVES STEM CELL RETENTION AND CELL-MEDIATED CARDIAC REPAIR

SINGLE CELL ENCAPSULATION IMPROVES STEM CELL RETENTION AND CELL-MEDIATED CARDIAC REPAIR

FUNCTIONAL ENHANCEMENT OF CIRCULATING ANGIOGENIC CELLS CULTURED ON A COLLAGEN BIOMATERIAL USING THE CCN1 MATRICELLULAR PROTEIN

Circulating angiogenic cells (CACs) play an important role in vascular homeostasis and hold tremendous promise for cell therapy for cardiovascular diseases. The regenerative potential of these cells can be improved by culture on a natural-based collagen matrix through the up-regulation of key integrin proteins. Further improvements are needed however, as the effects remain minimal or transient. The aim of this study was to evaluate whether the incorporation of CCN1, a secreted pro-angiogenic matricellular protein of the CCN (CYR61/CTGF/NOV) family, could further enhance the revascularization potential of CACs. Human CACs were cultured on fibronectin or a type I collagen matrix. Using RT-qPCR, CCN1 target integrins (Itg) αV and Itgβ3 were up-regulated 13.5±4.3 and 37.8±5.2 fold respectively, in CACs cultured on matrix compared to fibronectin. Using a CCN1-GFP protein, the association of CCN1 was 1.9±0.1 fold greater in CACs from matrix compared to fibronectin and this association was reduced by 41.5% and 42.6%, when an ItgαV or Itgβ3 blocking antibody was applied, respectively (p=0.008). Direct integrin interaction was confirmed by protein pull-down using a His-tagged CCN1 protein. Functionally, CCN1-matrix treatment increased proliferating Ki67+ cells 2.2± 0.2 fold over CACs on matrix alone (p=0.001) and 8.7±2.7 over fibronectin-cultured CACs (p=0.0002). When used in a migration assay, CCN1-matrix cells exhibited a 5.7±0.4 and 1.8±0.2 fold increase in migration towards vascular endothelial growth factor (VEGF) compared to fibronectin (p=0.001) and matrix (p=0.02) cultured cells, respectively. In a HUVEC/CAC angiogenic co-culture assay, CACs treated with the CCN1-matrix demonstrated a 4.1±1.6 and 7.3±1.4 fold increase in cell incorporation into capillary-like structures compared to matrix (p=0.03) and fibronectin (p=0.02) culture CACs. Tubule network formation during the angiogenic assay was also greater by 5.1±0.6 fold (p=0.007) for the CCN1-matrix treated CACs compared to matrix-cultured cells. In vivo, intramuscular injection of CCN1-matrix cultured CACs to ischemic hindlimbs of mice resulted in blood flow recovery to 80±1.8% of baseline, which was greater than the recovery observed for matrix-cultured CACs and PBS treatments (66±1.6% and 35.5±2.3%, respectively; p≤0.02). Matrix-cultured CACs were more responsive to CCN1 and exhibited greater angiogenic properties, suggesting that the specific integrin up-regulation may make the cells more responsive to extracellular signals. We also showed that pre-treatment of matrix-cultured CACs with CCN1 significantly improved their ability to restore perfusion in ischemic tissue. These findings demonstrate a novel mechanism which may be used to promote tissue neovascularization.

THREE DIMENSIONAL BLOOD AND CARDIAC STEM CELL SPHERES ENHANCE THE REGENERATIVE PERFORMANCE OF TRADITIONAL MONOLAYER CULTURE CELLS

Combination therapy with blood and cardiac stem cells after myocardial injury provide a complimentary paracrine profile that promotes angiogenesis and the generation of new myocardium. Given evidence that culture of cardiac stem cells (CSCs) as cardiospheres enhances regenerative performance, we test the hypothesis that growing both blood and cardiac stem cells as three dimensional spheres will recapitulate a stem cell niche environment to promote cell survival and cell-mediated repair. Human left atrial appendages and blood samples were obtained from patients undergoing clinically-indicated heart surgery. After seven days of monolayer culture, equivalent numbers of circulating angiogenic cells (CACs) and CSCs were combined in suspension culture which, over 7 days of culture, spontaneously formed spherical aggregates. Immunofluorescent lineage tracking demonstrated that both CACs and CSCs were heterogeneously distributed throughout the 50-80 uM spheres. CACs and CSCs grown together as spheres proliferated slowly as compared to monolayer culture of CSCs alone (2.0±0.4 fold less, p=0.01) or monolayer culture of CACs and CSCs together (1.8±0.18 fold less, p=0.02). Media conditioned under hypoxic, low serum conditions demonstrated that spherical culture did not inhibit cytokine production of angiogenin and VEGF while providing a 1.3±0.3 fold (n=4) increase in the production of the pro-survival cytokine SDF-1α. Consistent with these findings, media conditioned by CACs and CSCs after spherical culture promoted comparable degrees of HUVEC tubule formation when compared to media conditioned by monolayer culture of CACs and CSCs together (41.1±10.6 vs. 53.4±6.5 um respectively; p=0.36) while providing markedly greater recruitment of circulating stem cells (76.6±9.3 vs. 65.7±7.8 respectively; p=0.02). Implantation of sphered CACs and CSCs one week into immunodeficient mice after LAD ligation provided greater myocardial function relative to monolayer cultured CSCs (5±4 vs. 11±1% delta LVEF, p=0.04) with hints of superior regenerative performance when compared to traditional cardiospheres (9±1% delta LVEF, p=0.09). Spherical culture of CACs and CSCs together provides an incremental benefit towards cytokine production, recruitment of circulating stem cells and cell-mediated cardiac repair. This innovative culture technique may provide a means to directly apply stem cell therapy to the clinical setting when cell product yields are severely constrained.

Circulating angiogenic cell population responses to 10 days of reduced physical activity.

Circulating angiogenic cells (CACs) are a diverse group that have been identified as predictors of cardiovascular health and are inversely proportional to cardiovascular disease (CVD) outcomes. Inactivity is a growing concern in industrialized nations and is an independent risk factor for CVD. There is limited evidence regarding the impact of reduced physical activity (rPA) on different CAC populations. The purpose of this study was to evaluate the effect of objectively monitored rPA with maintained energy balance on two CAC populations (CFU and CD34(+) cells), intracellular nitric oxide (NOi), and genes related to NO production in active, healthy men. Participants (age 25 ± 2.9 yr) refrained from structured physical activity for 10 days, which was reflected by a significant reduction in time in vigorous + very vigorous intensity activity (P = 0.03). Sedentary time tended to increase (P = 0.06) with rPA. CFU CACs have been characterized as mainly monocytic and lymphocytic cells. We found significant reductions in both the number of CFU CACs (-35.69%, P = 0.01) and CFU CAC NOi (-33.84%, P = 0.03). Neither NOi nor the number of CD34(+) cells, which are hematopoietic and endothelial progenitors, changed with rPA. We found no significant differences in NO-related gene expression or oxidative stress-related gene expression with rPA in either CAC type. Therefore, we conclude that although various CAC populations have been related to vascular health, regular physical activity is necessary to maintain CAC NOi and the vulnerability of CACs to short-term reductions in physical activity is population specific.

Characterization of a degradable polar hydrophobic ionic polyurethane with circulating angiogenic cells in vitro

This study investigated the interaction of human circulating angiogenic cells (CACs) with a degradable polar hydrophobic ionic polyurethane (D-PHI) which has been previously shown to exhibit anti-inflammatory character and favorable interactions with human endothelial cells (ECs). Given the implication of the CACs in microvessel development it was of intrinsic interest to expand our knowledge of D-PHI biocompatibility with this relevant primary cell involved in angiogenesis. The findings will be compared to a well-established benchmark substrate for CACs, fibronectin-coated tissue culture polystyrene (TCPS). Immunoblotting analysis showed that CACs were a heterogeneous population of cells composed mostly of monocytic cells expressing the CD14 marker. Assessment of the cytokine release profile, using ELISA, showed that D-PHI supported a higher concentration of interleukin-10 (IL-10) when compared to the concentration of tumor necrosis factor alpha, which is indicative of an anti-inflammatory phenotype, and was different from the response with TCPS. It was found that the CACs were attached to D-PHI and remained viable and functional (nitric oxide production) during the seven days of culture. However, there did not appear to be any significant proliferation on D-PHI, contrary to the CAC growth on fibronectin-coated TCPS. It was concluded that D-PHI displayed some of the qualities suitable to enable the retention of CACs onto this substrate, as well as maintaining an anti-inflammatory phenotype, characteristics which have been reported to be important for angiogenesis in vivo.