Endothelial progenitor cells in vascular remodeling

### Endothelial Progenitor Cells and Postnatal Vasculogenesis: Experimental Evidence The option of performing full-scale endothelial cell transplantation to optimize local neovascularization is daunting if even feasible. An alternative, attractive strategy is designed to exploit the conceptual notion that endothelial cells and hematopoietic stem cells were ultimately derived from a common precursor, the putative hemangioblast. Hematopoietic stem cells had been shown previously to be present in circulating blood, in quantities sufficient to permit their harvesting and readministration for autologous, in lieu of bone marrow, transplantation. The related descendants, endothelial progenitor cells, can be detected in the peripheral circulation.1,2 Initially, Flk-1 and a second antigen, CD34, shared by angioblasts and hematopoietic stem cells were used to isolate putative angioblasts from the leukocyte fraction of peripheral blood.1 Meanwhile, endothelial progenitor cells were isolated from human umbilical cord blood,3 bone marrow–derived mononuclear cells,4 and CD34+ or CD133+ hematopoietic stem cells1,5 and were successfully ex vivo expanded with the use of human peripheral blood mononuclear cells.6 These cells differentiated into endothelial cells, as shown by expression of various endothelial proteins (KDR, von Willebrand factor, endothelial nitric oxide synthase, VE-cadherin, CD146) and uptake of Dil-acetylated LDL and binding of lectin.1,7 In animal models of ischemia, heterologous, homologous, and autologous endothelial progenitor cells were shown to incorporate into sites of active neovascularization in ischemic and tumor tissue. Blood flow recovery and capillary density were markedly improved, and the rate of limb loss was significantly reduced after transplantation of human peripheral blood–derived endothelial progenitor cells8,9 or bone marrow mononuclear cells.10 Likewise, infusion of peripheral blood–derived endothelial progenitor cells,11 bone marrow mononuclear cells,12 or purified CD34+ cells13 improved neovascularization and myocardial function after infarction. Isolated CD34+ cells also increased impaired blood flow in diabetic mice.14 These findings provide evidence that exogenously …

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

See related article, pages 536–544 Endothelial progenitor cells (EPCs) were introduced to a broad scientific readership in 1997 by Asahara et al, who demonstrated that CD34+ cells from the peripheral blood can adopt an endothelial cell-like phenotype in vitro.1 This culture-modified cell type (subsequently also termed endothelial outgrowth cells) improved ischemic neovascularization after intravenous transfusion.2 The prospect of ameliorating tissue ischemia by ex vivo–expanded autologous angioblasts resulted in extensive research activities, including the therapeutic application in patients with myocardial ischemia.3 However, the results are still conflicting, which is at least partially attributable to the fact that EPCs comprise a heterogenous pool of subpopulations originating from distinct sources and displaying diverse phenotypes.4 For instance, the common characterization of EPCs as CD34+CD133+VEGF-R2+ by flow cytometry has been recently questioned in different studies showing that only CD133−CD45− cells differentiate into endothelial cells.5,6 Early outgrowth of endothelial-like cells from mononuclear cells cultured for 5 to 7 days in the presence of endothelial growth factors represent a monocyte-like subtype with low proliferative capacity secreting high …

To the Editor: In their very interesting paper, Rehman et al1 demonstrate that the vast majority of peripheral blood–derived acLDL+ Ulex-lectin+ cells in culture—often referred to as endothelial progenitor cells (EPC)—express monocyte/macrophage markers, indicating that they originate mainly from the monocyte/macrophage lineage. They suggest that only a small population of true stem cells/progenitor cells and endothelial cells exists that may originate directly from the hemangioblast or from hematopoietic stem cells. Our recent observations support their findings that the majority of EPC are derived from the monocyte/macrophage–containing CD34− mononuclear cell population. Nevertheless, we could also identify and quantify a minor proportion of EPC that has its origin in the hematopoietic stem …

endothelial progenitor cells (EPCs) are a unique cell type found circulating in the peripheral blood with the capacity to become mature endothelial cells. EPCs can be released from many sources including the bone marrow, adipose tissue, the vessel wall, as well as potentially the spleen, liver, and

The hemangioblast: Cradle to clinic

Alzheimer's disease (AD) is the main neurodegenerative disease leading to dementia and cognitive impairment in the elderly. Considering AD to be an epidemic, an increase from the current 50 million to more than 150 million patients is expected by the year 2050. AD is characterized by a slow, progressive and asymptomatic onset; making it difficult to decipher the precise etiology. It is well established that AD presents two characteristic features, extracellular β-amyloid plaques and intracellular tau tangles, that eventually lead to the impairment of cognitive functions. Unfortunately, AD symptomatology shares many similarities with other dementias once is present, which makes it difficult an accurate premortem diagnosis. Although AD is mainly considered an aging-related condition that affects cognitive function, several cardio- and cerebrovascular comorbidities such as hypertension or diabetes are also risk factors for cognitive impairment. Accordingly, brain vascular-associated alterations underlie many pathophysiological mechanisms of AD. We have recently reviewed the latest evidence supporting the detrimental role of vascular and angiogenic alterations during AD (Custodia et al., 2022). Remarkably, cerebral blood-brain barrier (BBB) leakage and microbleeds are associated with cognitive decline in patients with mild cognitive impairment (MCI) and early AD. Accordingly, the two-hit vascular hypothesis points at initial damage in cerebral vasculature (hit one) that eventually induces the accumulation of β-amyloid (Aβ) in the brain (hit two; Zlokovic, 2005). CD34+ bone marrow-derived progenitor cells (BMPCs) define a group of stem and progenitor cell populations released by the bone marrow that covers different subpopulations of cells from the hematopoietic linage, including endothelial progenitor cells (EPCs). EPCs exhibit characteristics of both endothelial and stem cells, and, accordingly, proangiogenic early EPCs expressing both CD34 and CD133 (a progenitor surface marker) can be distinguished from late EPCs additionally expressing KDR and/or CD146 (endothelial markers), which participate in the process of angiogenesis and vasculogenesis (Figure 1). Therefore, EPCs participate in angiogenesis and the maintenance of the endothelium by acting as a cellular reservoir for the replacement of dysfunctional endothelial cells, or by the secretion of angiogenic growth factors.Figure 1: Beneficial roles of CD34+ BMPCs following CNS injury.CD34+ BMPCs and the EPCs subtypes, early and late, can promote both angiogenesis and vasculogenesis following CNS injury by specializing in endothelial cells, and/or indirectly by secreting free and exosome-enveloped growth factors. G-CSF is a glycoprotein that acts in the bone marrow to mobilize both EPCs and CD34+ BMPCs after damage. BMPCs: Bone marrow progenitor cells; CNS: central nervous system; EPCs: endothelial progenitor cells; G-CSF: granulocyte colony-stimulating factor. Created with BioRender.com.Given that both dysfunctional angiogenesis and compromised BBB integrity seem critical in the onset and/or progression of AD, CD34+ progenitor cells, primarily EPCs, appear as potential targets for the early diagnosis and/or treatment of the disease. In this way, early and late EPCs would work synergistically: early EPCs reach the site of injury due to the high concentration of angiogenic factors and inflammatory cytokines, from which they paracrinally release different factors promoting angiogenesis and recruiting late EPCs, which either restore the endothelium or form new vessels guided by the early EPCs. Here, we discuss recent work and ongoing human clinical trials testing the feasibility of CD34+ BMPCs and EPCs as early biomarkers of AD and pharmacological targets for future treatments. Association of circulating levels of CD34+ BMPCs and cognitive decline in healthy and MCI subjects: Several cross-sectional studies have shown that the number of circulating CD34+ BMPCs decreases with age, and this may impact cognition. In this regard, a longitudinal study regarding cognition and CD34+ BMPCs levels reported that older healthy subjects had lower levels of CD34+ BMPCs than younger counterparts at baseline measurements (Hajjar et al., 2016). Moreover, this investigation revealed that subjects with higher baseline levels of several subgroups of CD34+ BMPCs such as early and late EPCs, and CD34+/KDR+ cells, among others, had better executive-derived and working memory scores over 4 years of follow-up (Hajjar et al., 2016). Recently, a large transverse study has shown the association between CD34+ BMPCs and different memory-related tests in cognitively normal subjects with coronary artery disease (Moazzami et al., 2020). Notably, circulating numbers of late EPCs were positively correlated with a better performance in tasks assessing visual, logical, and verbal immediate/delayed memory. Therefore, the amount of circulating CD34+ BMPCs subtypes appears to be negatively correlated to the cognitive decline of both healthy subjects and patients with vascular-associated conditions. Although more longitudinal clinical studies are needed to fully confirm the harmful effect of low levels of CD34+ BMPCs on the cognitive state, and other factors may be also taking part in this cognitive decline, it is still plausible that larger amounts of circulating endothelial progenitors exert a protective effect, probably by the maintenance of vascular endothelium integrity. MCI often precedes clinical symptoms of AD, and MCI patients show an increased risk of developing dementia in the future. Thus, it is very interesting to study CD34+ BMPCs/EPCs levels in patients with MCI in order to test whether such levels can be used as potential non-invasive diagnostic biomarkers to detect cognitive decline or its progression from MCI to dementia. Some studies have observed a decrease in CD34+ BMPCs and EPCs populations from MCI patients (Nation et al., 2018; Callahan et al., 2020). In this sense, MCI patients with lower levels of circulating CD34+ BMPCs and both subtypes of EPCs exhibited worse scores in memory tests and reduced cortical thickness compared to control subjects (Nation et al., 2018). Considering the angiogenesis ratio (pro-angiogenic/non-angiogenic BMPCs, including early and late EPCs), Callahan et al. (2020) showed a positive association between angiogenesis ratio and white matter hyperintensities, but not with global cerebral blood flow, hippocampal volume, or accumulation of tau and Aβ. By contrast, measurements in an older cohort of MCI patients did not show significant changes in CD34+, early EPCs, and late EPCs circulating levels compared to control subjects (Breining et al., 2016). This discrepancy may highlight that aging decreases CD34+ BMPCs to such a reduced level that is no longer different in controls compared to MCI. In summary, it seems that the reduction in CD34+ BMPCs is directly related to vascular dysfunction, increasing brain white matter microlesions and impairing cognition in MCI patients. Association of circulating levels of CD34+ BMPCs and AD: Several studies have been performed in order to determine the relationship between CD34+ BMPCs/EPCs circulating levels and the progression of AD (Maler et al., 2006; Lee et al., 2009; Stellos et al., 2010; Bigalke et al., 2011; Kong et al., 2011; Breining et al., 2016; Callahan et al., 2020; Haiyuan et al., 2020). In this way, AD patients in the early symptomatic phase already showed lower levels of CD34+ and CD34+/KDR+ cells compared to their control counterparts (Maler et al., 2006; Haiyuan et al., 2020). Notably, CD34+ BMPCs counts have negatively correlated with levels of Aβ1–42 in cerebrospinal fluid and the Aβ ratio 42/40, two well-known biomarkers for AD, as well as with age, only in the early AD group (Maler et al., 2006). Furthermore, the homing capacity of EPCs from early AD patients was already impaired (Haiyuan et al., 2020). Overall, it is becoming clear that dysfunctional CD34+ BMPCs are related to a reduced ability to repair brain endothelial cells, which appears to mediate neurotoxicity by affecting the BBB permeability. On the other hand, different outcomes were described in studies assessing the number of progenitor cells during AD progression. Specifically, lower counts of CD34+ BMPCs and EPCs have been observed in moderate and severe AD patients compared to both early AD stage (Haiyuan et al., 2020) and control subjects (Lee et al., 2009; Kong et al., 2011; Haiyuan et al., 2020). Such studies also revealed that homing and adhesion features of EPCs from AD patients were impaired (Haiyuan et al., 2020), as well as EPCs levels were inversely correlated with the mini-mental state exam (MMSE) score (Lee et al., 2009; Stellos et al., 2010; Kong et al., 2011). Furthermore, moderate to severe AD patients displayed a reduced flow velocity of the middle cerebral artery (Kong et al., 2011). In contrast, other studies reported higher levels of CD34+ BMPCs and EPCs compared to controls (Stellos et al., 2010; Bigalke et al., 2011), or even no changes (Breining et al., 2016). Intriguingly, the work from Stellos and colleagues reported an increase in both CD34+ BMPCs and early EPCs counting when comparing moderate to severe AD patients versus control subjects; however, within the AD group, there was an inverse correlation between CD34+ BMPCs and early EPCs counting and the MMSE score. Although these results seem contrary to each other, it is noteworthy that most AD patients from this study (Stellos et al., 2010) were treated with cholinesterase inhibitor; a drug involved in EPCs proliferation. Therefore, this fact may bias the results and it could explain why cell counting in the AD group was higher than in controls, but they were inversely correlated with MMSE scores. The other study that showed increased levels of CD34+ BMPCs/EPCs (Bigalke et al., 2011) only measured the numbers of CD34+ BMPCs in early to moderate AD compared to controls, with no information regarding cholinesterase inhibitor treatment. In summary, most of the studies in later AD stages support the studies performed on MCI and early AD stages. Therefore, AD-mediated loss of CD34+ BMPCs/EPCs, as well as loss of EPCs-intrinsic features, are likely present in AD patients and may constitute novel diagnostic and therapeutic targets. Potential therapy with granulocyte colony-stimulating factor (G-CSF) in AD: The G-CSF is a glycoprotein secreted by endothelial and immune cells that acts as a hematopoietic growth factor (Figure 1). Among other beneficial mechanisms following vascular injury, the G-CSF can promote angiogenesis by mobilizing EPCs (Figure 1). Therefore, G-CSF may be a potential target to enhance vascular repair in AD patients. Indeed, it has been recently shown that a G-CSF treatment improved memory as well as reduced blood levels of amyloid and tau in mild to moderate patients of AD (Potter et al., 2021). Based on these achievements, it is currently conducting a phase2b clinical trial in order to evaluate the long-term treatment of G-CSF in AD patients (NCT04902703; ClinicalTrials.gov). It would be interesting to look at CD34+ BMPCs and EPCs levels from those clinical trials in order to elucidate whether such potential benefits promoted by G-CSF therapy are totally or partially mediated by increasing CD34+ BMPCs/EPCs mobilization. Future challenges: The body of evidence supporting a vascular component underlying AD onset and/or progression is growing. However, further studies are mandatory to elucidate whether such vascular component triggers AD, is a consequence of AD, or both. Moreover, longitudinal studies are needed to confirm the relationship between CD34+ BMPCs/EPCs levels and AD progression. Given that vascular-related diseases may influence the amount of circulating progenitor cells, especially in AD patients, comorbidities present in those subjects deserve special attention when interpreting the results. Likewise, pharmacological treatments, such as a cholinesterase inhibitor, may bias the results from studies giving uncorrected information. Despite the promising results in animal models of AD, the number of published results and clinical trials regarding the direct application of EPCs as a potential therapy in AD patients is absent. This is remarkable when there is compelling evidence that supports the role of endothelial dysfunction in the onset and progression of AD, and the potential of EPCs as a diagnostic biomarker and/or therapeutic target (Custodia et al., 2022). However, we were unable to find published data or ongoing clinical trials in humans using the application of EPCs to treat AD; as already seen in a stroke clinical trial (NCT01468064). Moreover, several recent studies have highlighted the beneficial role of EPCs secretome/exosomes by protecting and repairing the BBB following damage without using a cell-based therapy. So, clinical trials based on EPCs-derived secretome/exosomes might be a safer and more promising approach in AD research. Finally, only the GCSF-based treatment is being tested in AD patients at later stages, with modest but promising results. Given that endothelium-related impairments are already seen in MCI patients, it would be really interesting to test this GCSF-based treatment in those subjects in order to increase the benefits and protect against the progression to AD. This work was partially supported by grants from the Xunta de Galicia (IN607A2018/3 to TS, IN607D 2020/09 to TS, IN606A-2021/015 to AC; IN606B-2021/010 to DRS), and Science Ministry of Spain (RTI2018-102165-B-I00 to TS, RTC2019-007373-1 to TS). Furthermore, this work was also supported by grants from the INTERREG Atlantic Area (EAPA_791/2018_ NEUROATLANTIC project to TS), INTER-REG V A España Portugal (POCTEP) (0624_2IQBIONEURO_6_E to TS), and the European Regional Development Fund (ERDF). Moreover, DRS (CD21/00166) and TS (CPII17/00027) are recipients of research contracts from the Sara Borrell and Miguel Servet Programs, respectively, from the Instituto de Salud Carlos III. Availability of data and materials:All data generated or analyzed during this study are included in this published article and its supplementary information files. Open peer reviewers:Yali Jia, Beijing Institute of Radiation Medicine, China; Rongcan Luo, Kunming Institute of Zoology Chinese Academy of Sciences, China. Additional file:Open peer review reports 1 and 2.P-Reviewers: Jia Y, Luo R; C-Editors: Zhao M, Liu WJ, Wang Lu; T-Editor: Jia Y

Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells

pulmonary hypertension (PH), diagnosed when mean pulmonary arterial pressure exceeds the upper limits of normal (i.e., >25 mmHg) at rest (2), occurs in a variety of clinical situations and is associated with a broad spectrum of histological patterns and abnormalities. PH is currently classified into five distinct World Health Organization (WHO) groups, based on common clinical parameters, potential etiological mechanisms, and responses to treatment (22). Although any form of PH can contribute to increased patient morbidity and mortality, pulmonary arterial hypertension (PAH) (WHO group 1) is a particularly severe and progressive form associated with right heart failure and premature death (1). At present, therapeutic approaches to stabilize or reverse this debilitating condition involve treatment with one or a combination of up to three specific classes of agents, including prostacyclin analogs, endothelin-1 receptor antagonists, and/or phosphodiesterase-5 inhibitors. Retrospective (meta)analyses of these therapeutic strategies have demonstrated a reduction in mortality with their use (7, 12); however, many experts believe that current PAH treatment is inadequate given the persistently high mortality rate and functional hemodynamic impairment in many patients. These observations have led to continued intensive investigation into pathogenetic mechanisms and many proposals for additional alternative new therapies (20, 24). Among the potential new therapies, increasing interest in the role of endothelial progenitor cells (EPCs) as a cell-based therapy has emerged. However, issues remain regarding what group of PH patients are most likely to benefit from treatment, at what point in the disease is treatment most likely to be successful, and what types of cells should be utilized for therapy.

Readers of Arteriosclerosis, Thrombosis, and Vascular Biology are already well aware of the high level of interest in gene therapy as a potential means of combating cardiac and vascular disease. During the last 10 years or so there have been clear demonstrations in a variety of animal models that it is possible to modulate the progress of atherosclerotic vascular disease, restenotic arterial disease, or venous graft disease by transgenic or knockout approaches. These studies have concomitantly generated a series of experiments in which local gene therapy, predominantly using adenoviral vectors, has been used to attempt to ameliorate restenosis in grafts or after vascular injury and to enhance angiogenesis in and around areas of ischemic or infarcted tissue. The essential vision of successful human gene therapy for cardiovascular diseases remains undimmed,1 but the original enthusiastic optimism has been tempered by reality: there are significant generic problems such as inflammatory side effects due to the current generation of viral vectors and potential safety concerns for long-term gene therapy highlighted by the cases of leukemia in children being treated for immunodeficiency diseases2 in addition to the complications inherent in trying to treat complex disease processes with a single magic bullet. Nonetheless, several uncontrolled clinical trials, but very few controlled trials so far, have suggested benefit from local transfer of the gene for vascular endothelial growth factor into the heart after myocardial infarction or into the leg to overcome peripheral ischemia.3,4 See page 2266 While the significant hurdles that need to be overcome to make cardiovascular gene therapy a clinical …

No abstract available

Tissue replacement in the adult organism by cell-specific differentiation of autologous stem/progenitor cells has evolved as a fascinating concept in stem cell biology. After organ damage, bone marrow–derived circulating or tissue-resident progenitor cells are thought to differentiate toward the type of cell needed for repair. According to this concept, maturation of these cells would be expected to result at best in a perfect morphological and functional replacement of the injured tissue. However, in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , He et al show that endothelial progenitor cells (EPCs) are more than just as capable of in vitro angiogenic tube formation as mature endothelial cells (ECs), but are truly advantageous when it comes to stress tolerance.1 See page 2021 EPCs were originally characterized as cells that are mobilized from the bone marrow and circulate in the peripheral blood and express certain surface membrane markers including the vascular endothelial growth factor receptor (VEGF-R2) KDR and the hematopoietic progenitor cell markers CD34 and CD133.2–4 During ex vivo expansion, these cells develop morphological and functional characteristics typical for ECs, including formation of vascular-like structures in matrigel and other in vitro angiogenesis assays. Most importantly, however, transplanted EPCs exhibit an extraordinary potent in vivo capacity to improve the neovascularization of ischemic tissue in the adult organism.5 In this regard, EPCs were shown to be more effective than mature ECs in animal models of hind limb ischemia,6–8 although mature ECs are well established to exert a potent in vitro angiogenic activity. Thus, the …

To the Editor: Crosby et al1 noted in their report that the recruitment of bone marrow–derived endothelial progenitor cells to newly forming blood vessels might have been hitherto overlooked. Postulated already a century ago,2 there is now ample evidence for a close association between blood progenitor cells and angiogenesis3 and the existence of a hemangioblastic progenitor capable …

Angiogenesis and vasculogenesis: Inducing the growth of new blood vessels and wound healing by stimulation of bone marrow–derived progenitor cell mobilization and homing

To the Editor: Rapid (re)endothelialization of autologous and synthetic vascular grafts is regarded as a key element in reducing the risk of acute thrombosis and, by limiting adverse vessel wall remodeling, which includes intimal hyperplasia, preserving long-term patency. Rotmans et al1 (and the accompanying editorial2) challenge this tenet of vascular biology in their report of human anti-CD34 antibody–coated polytetrafluoroethylene (PTFE) grafts in a porcine AV fistula model. Rapid “endothelialization” of grafts is described, which, rather than improving functional outcome, was associated with significantly increased intimal hyperplasia at the venous anastomosis. The authors conclude that future studies will need to address a putative deficiency in the functional capability of captured endothelial progenitor cells (EPCs), highlighting approaches based on the use of regulatory proteins or alternative cell combinations. In our opinion, the data do not support these conclusions. The graft endoluminal cells were inadequately characterized with lectin (a surface antigen shared by several blood-borne …