Resident Endothelial Progenitor Cells Research Articles

MTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

Towards Novel Geneless Approaches for Therapeutic Angiogenesis.

Cardiovascular diseases are the leading cause of mortality worldwide. Such a widespread diffusion makes the conditions affecting the heart and blood vessels a primary medical and economic burden. It, therefore, becomes mandatory to identify effective treatments that can alleviate this global problem. Among the different solutions brought to the attention of the medical-scientific community, therapeutic angiogenesis is one of the most promising. However, this approach, which aims to treat cardiovascular diseases by generating new blood vessels in ischemic tissues, has so far led to inadequate results due to several issues. In this perspective, we will discuss cutting-edge approaches and future perspectives to alleviate the potentially lethal impact of cardiovascular diseases. We will focus on the consolidated role of resident endothelial progenitor cells, particularly endothelial colony forming cells, as suitable candidates for cell-based therapy demonstrating the importance of targeting intracellular Ca2+ signaling to boost their regenerative outcome. Moreover, we will elucidate the advantages of physical stimuli over traditional approaches. In particular, we will critically discuss recent results obtained by using optical stimulation, as a novel strategy to drive endothelial colony forming cells fate and its potential in the treatment of cardiovascular diseases.

Impact of Glucose-Lowering Medications on Cardiovascular and Metabolic Risk in Type 2 Diabetes.

Type 2 Diabetes Mellitus (T2DM) is associated with a high risk of atherosclerotic cardiovascular (CV) disease. Among the well-known pathophysiologic factors, crucial roles are played by endothelial dysfunction (caused by oxidative stress and inflammation hyperglycemia-linked), increased activity of nuclear factor kB, altered macrophage polarization, and reduced synthesis of resident endothelial progenitor cells. As consequence, a potentially rapid progression of the atherosclerotic disease with a higher propensity to unstable plaque is arguable, finally leading to significantly increased cardiovascular mortality. Main managements are focused on both prevention and early diagnosis, by targeted treatment of hyperglycemia and vascular complications. Innovative therapeutic approaches for T2DM seek to customize the antidiabetic treatment to each patient in order to optimize glucose-lowering effects, minimize hypoglycemia and adverse effects, and prevent cardiovascular events. The newer drugs (e.g., Glucagon Like Peptide-1 Receptor Agonists, GLP-1 RAs; Sodium GLucose coTransporter-2 inhibitors, SGLT2is; DiPeptidyl Peptidase-4 inhibitors, and DPP4is) impact body weight, lipid parameters, and blood pressure, as well as endothelial (dys)functions, inflammatory markers, biomarkers of both oxidative stress, and subclinical atherosclerosis. The present review summarizes the results of the main trials focused on the cardiovascular safety of these drugs from the CV standpoint.

PKM2 regulates angiogenesis of VR-EPCs through modulating glycolysis, mitochondrial fission, and fusion.

Vascular resident endothelial progenitor cells (VR-EPCs) have a certain ability to differentiate into endothelial cells (ECs) and participate in the process of angiogenesis. Glycolysis and mitochondrial fission and fusion play a pivotal role in angiogenesis. Pyruvate kinase muscle isoenzyme 2 (PKM2), which mediates energy metabolism and mitochondrial morphology, is regarded as the focus of VR-EPCs angiogenesis in our study. VR-EPCs were isolated from the hearts of 12-weeks-old Sprague-Dawley rats. The role of PKM2 on angiogenesis was evaluated by tube formation assay, wound healing assay, transwell assay, and chick chorioallantoic membrane assay. Western blot analysis, flow cytometry, mitochondrial membrane potential detection, reactive oxygen species (ROS) detection, immunofluorescence staining, and quantitative real-timepolymerase chain reaction were used to investigate the potential mechanism of PKM2 for regulating VR-EPCs angiogenesis. We explored the function of PKM2 on the angiogenesis of VR-EPCs. DASA-58 (the activator of PKM2) promoted VR-EPCs proliferation and PKM2 activity, it also could promote angiogenic differentiation. At the same time, DASA-58 significantly enhanced glycolysis, mitochondrial fusion, slightly increased mitochondrial membrane potential, and maintained ROS at a low level. C3k, an inhibitor of PKM2, inhibited PKM2 activity, expression of angiogenesis-related genes and tube formation. Besides, C3k drastically reduced glycolysis and mitochondrial membrane potential while significantly promoting mitochondrial fission and ROS level. Activation of PKM2 could promote VR-EPCs angiogenesis through modulating glycolysis, mitochondrial fission and fusion. By contrast, PKM2 inhibitor has opposite effects.

Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis.

Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo.

Hypoxia-induced proliferation of tissue-resident endothelial progenitor cells in the lung.

Exposure to hypoxia induces changes in the structure and functional phenotypes of the cells composing the pulmonary vascular wall from larger to most peripheral vessels. Endothelial progenitor cells (EPCs) may be involved in vascular endothelial repair. Resident EPCs with a high proliferative potential are found in the pulmonary microcirculation. However, their potential location, identification, and functional role have not been clearly established. We investigated whether resident EPCs or bone marrow (BM)-derived EPCs play a major role in hypoxic response of pulmonary vascular endothelial cells (PVECs). Mice were exposed to hypoxia. The number of PVECs transiently decreased followed by an increase in hypoxic animals. Under hypoxic conditions for 1 wk, prominent bromodeoxyuridine incorporation was detected in PVECs. Some Ki67-positive cells were detected among PVECs after 1 wk under hypoxic conditions, especially in the capillaries. To clarify the origin of proliferating endothelial cells, we used BM chimeric mice expressing green fluorescent protein (GFP). The percentage of GFP-positive PVECs was low and constant during hypoxia in BM-transplanted mice, suggesting little engraftment of BM-derived cells in lungs under hypoxia. Proliferating PVECs in hypoxic animals showed increased expression of CD34, suggesting hypoxia-induced gene expression and cell surface antigen of EPC or stem/progenitor cells markers. Isolated PVECs from hypoxic mice showed colony- and tube-forming capacity. The present study indicated that hypoxia could induce proliferation of PVECs, and the origin of these cells might be tissue-resident EPCs.

Circulating and tissue resident endothelial progenitor cells

Progenitor cells for the endothelial lineage have been widely investigated for more than a decade, but continue to be controversial since no unique identifying marker has yet been identified. This review will begin with a discussion of the basic tenets originally proposed for proof that a cell displays properties of an endothelial progenitor cell. We then provide an overview of the methods for putative endothelial progenitor cell derivation, expansion, and enumeration. This discussion includes consideration of cells that are present in the circulation as well as cells resident in the vascular endothelial intima. Finally, we provide some suggested changes in nomenclature that would greatly clarify and demystify the cellular elements involved in vascular repair.

Clinically Relevant Microfluidic Magnetophoretic Isolation of Rare-Cell Populations for Diagnostic and Therapeutic Monitoring Applications

Cells of biomedical interest are, despite their functional significance, often present in very small numbers. Therefore the analysis and isolation of previously inaccessible rare cells, such as peripheral hematopoietic stem cells, endothelial progenitor cells, or circulating tumor cells, require efficient, sensitive, and specific procedures that do not compromise the viability of the cells. The current study builds on previous work on a rationally designed microfluidic magnetophoretic cell separation platform capable of throughputs of 240 μL min(-1). Proof-of-concept was first conducted using MCF-7 (1-1000 total cells) as the target rare cell spiked into high concentrations of Raji B-lymphocyte nontarget cells (~10(6) total cells). These experiments lead to the establishment of a magnet-based separation for the isolation of 50 MCF-7 cells directly from whole blood. Results show an efficiency of collection greater than 85%, with a purity of over 90%. Next, resident endothelial progenitor cells and hematopoietic stem cells are directly isolated from whole human blood in a rapid and efficient fashion (>96%). Both cell populations could be simultaneously isolated and, via immunofluorescent staining, individually identified and enumerated. Overall, the presented device illustrates a viable separation platform for high purity, efficient, and rapid collection of rare cell populations directly from whole blood samples.

Identification of CD133-, CD34- and KDR-positive cells in the bovine ovary: A new site of vascular wall resident endothelial progenitor cells

Intense angiogenesis, vascular remodelling as well as regression of its vasculature are prerequisites for ovarian function with its cyclically developing and regressing follicles and corpora lutea. So far neither a stringent explanation for the enormous angiogenic potential of the ovary nor its cellular origins have been suggested. In an earlier study of our work group, endothelial cells were isolated from the bovine corpus luteum and cultivated in vitro. They performed vasulogenesis in vitro and showed properties of progenitor cells. The present study aimed at in situ identification of endothelial progenitor cells (EPCs) in the bovine ovary. Immunohistochemical examinations, based on the detection of KDR and CD34 co-labelled cells - a marker combination that amongst others is commonly accepted as typical for EPC identification - were performed. Hormonal cycle dependent expression varieties were analysed by the measurement of mRNA amounts of CD34 and KDR as well as the stem cell marker CD133 (Prominin-1). Ovarian samples comprising corpora lutea of varying stages (developing and mature corpus luteum, corpus luteum in regression, corpus luteum of pregnancy) from 17 adult cows were examined. Results show that specific mRNA of CD133, CD34 and KDR was expressed in ovaries of all luteal stages. Expression data analysis revealed significant differences in CD133 and CD34 expression levels between the luteal stages but no significant differences in KDR expression. CD34/KDR co-immunoreactive cells were predominantly situated within the media of arterial vessel wall. The detection of ovarian EPCs represents an important step towards further understanding of the mechanisms involved in the reproductive biology and pathophysiology of the ovary.

Progenitor Cells in the Pulmonary Circulation

The mechanisms involved in the regulation of pulmonary vascular endothelial cells to replace aged or injured cells remains poorly understood, although differences in proliferative potential between the microvascular and macrovascular endothelium are well described. The presence of resident pulmonary vascular endothelial progenitor cells in rats and mice has been recently reported. These resident endothelial progenitor cells display clonal proliferative potential, restricted expression of cell surface molecules to those typical of lung endothelium, and in vivo vessel-forming ability upon transplantation into recipient animals. The rat pulmonary microvascular endothelium is enriched in resident progenitor cells, with the highest proliferative potential compared with the pulmonary macrovascular endothelium. Preliminary evidence suggests that resident endothelial progenitor cells are present in the human lung vasculature, but whether differences in enrichment of the progenitors in various pulmonary vascular beds exist remains to be determined.

Resident Endothelial Progenitor Cells From Human Placenta Have Greater Vasculogenic Potential Than Circulating Endothelial Progenitor Cells From Umbilical Cord Blood.

Endothelial colony-forming cells (ECFCs) isolated from umbilical cord blood (CBECFCs) are highly proliferative and form blood vessels in vivo. The purpose of this investigation was to isolate and characterize a population of resident ECFCs from the chorionic villi of term human placenta and provide a comparative analysis of their proliferative and vasculogenic potential with CBECFCs. ECFCs were isolated from umbilical cord blood and chorionic villi from placentas obtained by caesarean deliveries. Placental ECFCs (PECFCs) expressed CD144, CD31, CD105, and KDR and were negative for CD45 and CD34, consistent with other ECFC phenotypes. PECFCs were capable of 28.6 ± 6.0 population doublings before reaching senescence (vs. 47.4 ± 3.2 for CBECFCs, p < 0.05, n = 4). In single cell assays, 46.5 ± 1.2% underwent at least one division (vs. 51.0 ± 1.8% of CBECFCs, p = 0.07, n = 6), and of those dividing PECFCs, 71.8 ± 0.9% gave rise to colonies of >500 cells (highly proliferative potential clones) over 14 days (vs. 69.4 ± 0.7% of CBECFCs, p = 0.07, n = 9). PECFCs formed 5.2 ± 0.8 vessels/mm(2) in collagen/fibronectin plugs implanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, whereas CBECFCs formed only 1.7 ± 1.0 vessels/mm(2) (p < 0.05, n = 4). This study demonstrates that circulating CBECFCs and resident PECFCs are identical phenotypically and contain equivalent quantities of high proliferative potential clones. However, PECFCs formed significantly more blood vessels in vivo than CBECFCs, indicating that differences in vasculogenic potential between circulating and resident ECFCs exist.

The role of single cell derived vascular resident endothelial progenitor cells in the enhancement of vascularization in scaffold-based skin regeneration

Increasing evidence suggests that vascular resident endothelial progenitor cells (VR-EPCs) are present in several organs, playing an important role in postnatal neovascularization. Here, we isolated and characterized VR-EPCs from cardiac tissue in vitro, evaluating their regenerative potential in vivo. VR-EPCs showed to be highly clonogenic and expressed several stem and differentiation markers. Under endothelial differentiation conditions, cells form capillary-like structures, in contrast to osteogenic or adipogenic differentiation conditions where no functional changes were observed. After seeding in scaffolds, cells were distributed homogeneously and directly attached to the scaffold. Then, cell seeded scaffolds were used to induce dermal regeneration in a nude mice full skin defect model. The presence of VR-EPCs enhanced dermal vascularization. Histological assays showed increased vessel number ( p < 0.05) and cellularization ( p < 0.05) in VR-EPCs group. In order to explore possible mechanisms of vascular regeneration, in vitro experiments were performed. Results showed that pro-angiogenic environments increased the migration capacity ( p < 0.001) and ability to form capillary-like structures ( p < 0.05) of VR-EPC. In addition, VR-EPCs secreted several pro-angiogenic molecules including VEGF and PDGF. These results indicate that a highly clonogenic population of VR-EPCs might be established in vitro, representing a new source for therapeutic vascularization in tissue engineering and regeneration.

High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells

Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.

CD40 activation is angioproliferative in pulmonary microvascular endothelial cells

IntroductionEndothelial CD40 is important during inflammation as an angiogenic stimulus. We tested whether CD40 expression in PMVEC is associated with angioproliferative potential.MethodsWestern analysis for CD40 expression was performed. Growth curves, barrier measurements, and scratch‐wound assays were carried out using an ECIS apparatus (Applied Biophysics) and a digital image system composed of an inverted microscope.ResultsBasal CD40 expression was high in PMVEC, whereas expression was absent in rat pulmonary arterial‐derived EC (PAEC). CD40 expression could be up‐regulated in PAEC following over‐expression of NAP‐1, an endothelial progenitor cell‐associated protein. Exposure of PMVEC to sCD40L (1 μg/mL) for 1 ‐ 48 hours increased proliferation and improved barrier integrity. Exposure to sCD40L also produced an accelerated PMVEC migration response with decreased wound closure times. These effects were inhibited by pre‐treatment with anti‐CD40 antibodies.ConclusionsOur data indicate activation of PMVEC‐expressed CD40 results in angioproliferative responses. These responses may be inherent to cell phenotypes expressing NAP‐1. Identification of PMVEC expressing CD40 in situ may localize pulmonary resident endothelial progenitor cells. Support: Parker B. Francis Foundation and AHA (0765421B) to T.M.M.

Lung microvascular resident endothelial progenitor cells exhibit high vasculogenic capacity

We recently cloned endothelial progenitor cells (EPCs) residing within the pulmonary microcirculation. However, it is not clear whether resident EPCs display de novo vessel formation, a sine qua non behavior of endothelial cells. We hypothesized that resident lung EPCs possess high vasculogenic capacity. Rat endothelial cells were isolated from the pulmonary artery (PAECs) and the pulmonary microcirculation (PMVECs), while resident EPCs were selected from PMVECs using a single cell clonogenic assay. An in vitro Matrigel assay revealed that PAECs organize in scattered patches, while PMVECs and EPCs form extensive, time-dependent networks beginning as early as 8 hours post-seeding (2, 7 and 8 networks/mm2, respectively). Vasculogenic capacity was determined by the de novo vessel formation within in vivo gel implants containing GFP-retrovirally transfected cells. Removal of plugs after 4 days demonstrated that PMVEC and EPCs, but not PAECs, form de novo vessels. After 10 days, EPCs display high vasculogenic capacity compared to PAECs and PMVECs (11, 3, and 8 vessels/mm2, respectively). Ultrastructural analysis revealed integrity of newly formed vessels. Immunostaining confirmed the presence of GFP-tagged cells within the intima of de novo vessels. The data indicate that lung microvascular EPCs display a uniquely high vasculogenic capacity, which is a critical determinant of vascular repair. HL66299.

Potential implications of vascular wall resident endothelial progenitor cells

A rapidly increasing body of data suggests an essential role of endothelial progenitor cells (EPCs) in vascular regeneration, formation of new vessels in cardiovascular diseases and also in tumor vasculogenesis. Moreover, recent data obtained from clinical studies with anti-angiogenic drugs in tumor therapy or with pro-angiogenic stimuli in ischemic disorders implicate a predictive role of the number of EPCs circulating in the peripheral blood in monitoring of these diseases. However, there is still some controversial data regarding the relevance of the EPCs in vascular formation depending on models used and diseases studied. One of the essential prerequisites for a better understanding of the whole contribution of EPCs to vascular formation in adult, a process called postnatal vasculogenesis, is to identify their exact sources. We could recently discover the existence of EPCs in a distinct zone of the vascular wall of large and middle sized adult blood vessels and showed that these cells are capable to differentiate into mature endothelial cells, to form capillary sprouts in arterial ring assay and to build vasa vasorum-like structures within the vascular wall. They also can be mobilized very rapidly from the vascular wall by tumor cells. This review will discuss the functional implications of these vascular wall resident endothelial progenitor cells (VW-EPCs) in relation to those of EPCs circulating in peripheral blood or derived from the bone marrow in cardiovascular and neoplastic diseases.