Induction of EPC homing on biofunctionalized vascular grafts for rapid in vivo self-endothelialization — A review of current strategies

For years intensive research has been done to improve the hemocompatibility of blood contacting vascular devices. Despite the enormous progress in physicochemical surface optimization technologies, the native endothelium still represents the ideal surface for blood contact. Numerous tissue engineering strategies aspired towards the endothelialization of graft surfaces to generate a non-thrombogenic barrier on artificial materials. A paradigm change in surface modification concepts is the in vivo endothelialization of vascular grafts by capturing circulating endothelial progenitor cells (EPCs) directly from the blood stream via biofunctionalized implant materials. Thereby, capture molecules are immobilized on artificial vascular grafts to mimic a pro-homing substrate for EPCs. In this review, different coating strategies for in vivo capturing of EPCs on synthetic implants are discussed. This therapeutic concept opens a new chapter in regenerative medicine by realizing the vision that every patient seeds his implants with his own progenitor cells to make the synthetic grafts unrecognizable for the body's rejection mechanisms.     

Renin-angiotensin system and vascular remodelling

The renin-angiotensin system (RAS) is compartmented between circulating blood and tissue pericellular space. Whereas renin and its substrate diffuse easily from one compartment to another, the angiotensin peptides act in the compartment where there are generated: blood or pericellular space. Renin is trapped in tissues by low and high affinity receptors. In the target cells, angiotensin II/AT1 receptor interaction generates different signals including an immediate functional calcium-dependent response, secondary hypertrophy and a late proinflammatory and procoagulant response. These late pathological effects are mediated by NADPH oxydase-generated free oxygen radicals and NFkappaB activation. In vivo, the tissue binding of renin and the induction of converting enzyme are the main determinants of the involvement of the RAS in vascular remodeling. The target cells of interstitial angiotensin II are mainly the vascular smooth muscle cells and fibroblasts, whereas the endothelial cells and circulating leukocytes are the main targets of circulating angiotensin II. In vivo, angiotensin II participates in the vascular wall hypertrophy associated with hypertension. In diabetes, as in other localized fibrotic cardiovascular diseases, the tissue effects of angiotensin II are mainly dependent on its ability to induce TGF-beta expression. In experimental atherosclerosis, angiotensin II infusion induces aneurysm formation mediated by activation of circulating leucocytes. In these models, the administration of angiotensin II antagonists has beneficial effects on pathological remodeling. Such beneficial effects of angiotensin II antagonists in localized pathological remodeling have not yet been demonstrated in humans.     

Alloimmunity to human endothelial cells derived from cord blood progenitors

There is considerable interest in exploiting circulating endothelial progenitor cells (EPCs) for therapeutic organ repair. Such cells may be differentiated into endothelial cells (ECs) in vitro and then expanded for use in tissue engineering. Vessel-derived ECs are variably immunogenic, depending upon tissue source, and it is unknown whether ECs derived from cord blood EPCs are able to initiate an allogeneic response. In this study, we compare the phenotype and alloantigenicity of human cord blood progenitor cell-derived ECs with HUVECs isolated from the same donors. Human cord blood progenitor cell-derived ECs are very similar to HUVECs in the expression of proteins relevant for alloimmunity, including MHC molecules, costimulators, adhesion molecules, cytokines, chemokines, and IDO, and in their ability to initiate allogeneic CD4(+) and CD8(+) memory T cell responses in vitro and in vivo. These findings have significant implications for the use of cord blood EPCs in regenerative medicine or tissue engineering.     

Pannexin channel and connexin hemichannel expression in vascular function and inflammation

Control of blood flow distribution and tissue homeostasis depend on the tight regulation of and coordination between the microvascular network and circulating blood cells. Channels formed by connexins or pannexins that connect the intra- and extracellular compartments allow the release of paracrine signals, such as ATP and prostaglandins, and thus play a central role in achieving fine regulation and coordination of vascular function. This review focuses on vascular connexin hemichannels and pannexin channels. We review their expression pattern within the arterial and venous system with a special emphasis on how post-translational modifications by phosphorylation and S-nitrosylation of these channels modulate their function and contribute to vascular homeostasis. Furthermore, we highlight the contribution of these channels in smooth muscle cells and endothelial cells in the regulation of vasomotor tone as well as how these channels in endothelial cells regulate inflammatory responses such as during ischemic and hypoxic conditions. In addition, this review will touch on recent evidence implicating a role for these proteins in regulating red blood cell and platelet function.     

Endothelial cells from cord blood CD133+CD34+ progenitors share phenotypic, functional and gene expression profile similarities with lymphatics

The existence of endothelial progenitor cells (EPC) with high cell-cycle rate in human umbilical cord blood has been recently shown and represents a challenging strategy for therapeutic neovascularization. To enhance knowledge for future cellular therapy, we compared the phenotypic, functional and gene expression differences between EPC-derived cells generated from cord blood CD34⁺ cells, and lymphatic and macrovascular endothelial cells (EC) isolated from human foreskins and umbilical veins, respectively. Under appropriate culture conditions, EPC developed into fully matured EC with expression of similar endothelial markers as lymphatic and macrovascular EC, including CD31, CD36, von Willebrand factor FVIII, CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-cadherin), Tie-1, Tie-2, VEGFR-1/Flt-1 and VEGFR-2/Flk-1. Few EPC-derived cells became positive for LYVE-1, indicating their origin from haematopoietic stem cells. However they lacked expression of other lymphatic cell-specific markers such as podoplanin and Prox-1. Functional tests demonstrated that the cobblestone EPC-derived cells up-regulated CD54 and CD62E expression in response to TNF-α, incorporated DiI-acetylated low-density liproprotein and formed cord- and tubular-like structures with capillary lumen in three-dimensional collagen culture – all characteristic features of the vascular endothelium. Structures compatible with Weibel-Palade bodies were also found by electron microscopy. Gene microarray profiling revealed that only a small percentage of genes investigated showed differential expression in EPC-derived cells and lymphatic EC. Among them were adhesion molecules, extracellular matrix proteins and cytokines. Our data point to the close lineage relationship of both types of vascular cells and support the theory of a venous origin of the lymphatic system.     

Deposition of Basic Fibroblast Growth Factor on Surface of Epidermal Melanocytes Suggesting the Stromal Control of Epidermal Pigmentation

Scanning electron microscopy with immunogold labeling was used to demonstrate the in vivo distribution of molecules of basic fibroblast growth factor (bFGF) that were expressed and/or present on the surface of the cells of the normal epidermis and dermal connective tissue of humans. We found that molecules of bFGF, seen as deposits of gold particles, were present densely on the surfaces of the melanocytes but not the epidermal keratinocytes. In connective tissue, these molecules were present exclusively on the surfaces of the fibroblasts, macrophages, vascular endothelial cells, and the basement membrane surrounding the endothelial tube. The selective deposition of bFGF molecules on the melanocytes suggests that the dermal connective tissue may be involved in controlling the proliferation of melanocytes by means of bFGF molecules in vivo, since these melanocytes require bFGF to proliferate in vitro. The latter is synthesized and stored exclusively in the connective tissue.     

Augmentation of Neovascularizaiton in Hindlimb Ischemia by Combined Transplantation of Human Embryonic Stem Cells-Derived Endothelial and Mural Cells

Background

We demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration.

Methods and Results

After the expansion of human VPC-derived vascular cells, we transplanted these cells to nude mice with hindlimb ischemia. The blood flow recovery and capillary density in ischemic hindlimbs were significantly improved in human VPC-derived EC-transplanted mice, compared to human peripheral and umbilical cord blood-derived endothelial progenitor cells (pEPC and uEPC) transplanted mice. The combined transplantation of human VPC-derived EC and MC synergistically improved blood flow of ischemic hindlimbs remarkably, compared to the single cell transplantations. Transplanted VPC-derived vascular cells were effectively incorporated into host circulating vessels as EC and MC to maintain long-term vascular integrity.

Conclusions

Our findings suggest that the combined transplantation of human ES cells-derived EC and MC can be used as a new promising strategy for therapeutic vascular regeneration in patients with tissue ischemia.     

Syk and Slp-76 mutant mice reveal a cell-autonomous hematopoietic cell contribution to vascular development

Developmental studies support a common origin for blood and endothelial cells, while studies of adult angiogenic responses suggest that the hematopoietic system can be a source of endothelial cells later in life. Whether hematopoietic tissue is a source of endothelial cells during normal vascular development is unknown. Mouse embryos lacking the signaling proteins Syk and Slp-76 develop abnormal blood-lymphatic endothelial connections. Here we demonstrate that expression of GFPSlp-76 in a subset of hematopoietic cells rescues this phenotype, and that deficient cells confer focal vascular phenotypes in chimeric embryos consistent with a cell-autonomous mechanism. Endogenous Syk and Slp-76, as well as transgenic GFPSlp-76, are expressed in circulating cells previously proposed to be endothelial precursors, supporting a causal role for these cells. These studies provide genetic evidence for hematopoietic contribution to vascular development and suggest that hematopoietic tissue can provide a source of vascular endothelial progenitor cells throughout life.     

Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth.     

Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung

How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy confirms this and shows that APP antibody targets nanoparticles to caveolae. Dynamic intravital fluorescence microscopy reveals that targeted caveolae operate effectively as pumps, moving antibody within seconds from blood across endothelium into lung tissue, even against a concentration gradient. This active transcytosis requires normal caveolin-1 expression. Whole body gamma-scintigraphic imaging shows rapid, specific delivery into lung well beyond that achieved by standard vascular targeting. This caveolar trafficking in vivo may underscore a key physiological mechanism for selective transvascular exchange and may provide an enhanced delivery system for imaging agents, drugs, gene-therapy vectors and nanomedicines. 'In vivo proteomic imaging' as described here integrates organellar proteomics with multiple imaging techniques to identify an accessible target space that includes the transvascular pumping space of the caveola.     

Endothelial progenitor cell sprouting in spheroid cultures is resistant to inhibition by osteoblasts: a model for bone replacement grafts

Survival of tissue transplants generated in vitro is strongly limited by the slow process of graft vascularization in vivo. A method to enhance graft vascularization is to establish a primitive vascular plexus within the graft prior to transplantation. Endothelial cells (EC) cultured as multicellular spheroids within a collagen matrix form sprouts resembling angiogenesis in vitro. However, osteoblasts integrated into the graft suppress EC sprouting. This inhibition depends on direct cell-cell-interactions and is characteristic of mature ECs isolated from preexisting vessels. In contrast, sprouting of human blood endothelial progenitor cells is not inhibited by osteoblasts, making these cells suitable for tissue engineering of pre-vascularized bone grafts.     

Establishment of outgrowth endothelial cells from peripheral blood

Blood outgrowth endothelial cells (BOECs) are important tools when investigating diagnostic and therapeutic approaches for vascular disease. In this protocol, mononuclear cells are isolated from peripheral blood and plated on type I collagen at ～135,000 cells per cm(2) in endothelial cell differentiation medium. On average, 0.34 colonies of endothelial cells per milliliter of blood can be obtained. Colonies of endothelial cells become visible after 14-28 d. Upon confluence, these rapidly expanding colonies can be passaged and have been shown to propagate up to 10(18)-fold. Isolated BOECs are phenotypically similar to vascular endothelial cells, as revealed by their cobblestone morphology, the presence of endothelial cell-specific Weibel-Palade bodies and the expression of endothelial cell markers such as VE-cadherin. The protocol presented here also provides a particularly useful tool for the ex vivo assessment of endothelial cell function from patients with different vascular abnormalities.     

Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy

Recent evidence suggests that the previously enigmatic cell type designated plasmacytoid monocytes can function as dendritic cells and contribute substantially to both innate and adaptive immunity. This cell type has previously been described only in bone marrow, blood, and organized lymphoid tissue, but not at effector sites with direct Ag exposure such as the mucosae. Plasmacytoid dendritic cells (P-DCs) matured in vitro can induce T cells to produce allergy-promoting Th2 cytokines; therefore, their possible occurrence in nasal mucosa during experimentally elicited allergic rhinitis was examined. Patients with silent nasal allergy were challenged topically with relevant allergen daily for 7 days. Biopsy specimens as well as blood samples were obtained before and during such provocation, and P-DCs were identified by their high expression of CD123 (IL-3R alpha-chain), together with CD45RA. Our results showed that P-DCs were present in low and variable numbers in normal nasal mucosa but increased dramatically during the allergic reaction. This accumulation concurred with the expression of the L-selectin ligand peripheral lymph node addressin on the mucosal vascular endothelium. The latter observation was particularly interesting in view of the high levels of L-selectin on circulating P-DC precursors and of previous reports suggesting that these cells can enter organized lymphoid tissue via high endothelial venules (which express peripheral lymph node addressin constitutively). Together, our findings suggested that P-DCs are involved in the triggering of airway allergy and that they are directed to allergic lesions by adhesion molecules that normally mediate leukocyte extravasation in organized lymphoid tissue.     

Expression, regulation, and function of atypical chemerin receptor CCRL2 on endothelial cells

Chemokine (CC motif) receptor-like 2 (CCRL2) binds leukocyte chemoattractant chemerin and can regulate local levels of the attractant, but does not itself support cell migration. In this study, we show that CCRL2 and VCAM-1 are upregulated on cultured human and mouse vascular endothelial cells (EC) and cell lines by proinflammatory stimuli. CCRL2 induction is dependent on NF-κB and JAK/STAT signaling pathways, and activated endothelial cells specifically bind chemerin. In vivo, CCRL2 is constitutively expressed at high levels by lung endothelial cells and at lower levels by liver endothelium; and liver but not lung EC respond to systemic LPS injection by further upregulation of the receptor. Plasma levels of total chemerin are elevated in CCRL2(-/-) mice and are significantly enhanced after systemic LPS treatment in CCRL2(-/-) mice compared with wild-type mice. Following acute LPS-induced pulmonary inflammation in vivo, chemokine-like receptor 1 (CMKLR1)(+) NK cell recruitment to the airways is significantly impaired in CCRL2(-/-) mice compared with wild-type mice. In vitro, chemerin binding to CCRL2 on endothelial cells triggers robust adhesion of CMKLR1(+) lymphoid cells through an α(4)β(1) integrin/VCAM-1-dependent mechanism. In conclusion, CCRL2 is expressed by EC in a tissue- and activation-dependent fashion, regulates circulating chemerin levels and its bioactivity, and enhances chemerin- and CMKLR1-dependent lymphocyte/EC adhesion in vitro and recruitment to inflamed airways in vivo. Its expression and/or induction on EC by proinflammatory stimuli provide a novel and specific mechanism for the local enrichment of chemerin at inflammatory sites, regulating the recruitment of CMKLR1(+) cells.     

Hydroxycarbamide Decreases Sickle Reticulocyte Adhesion to Resting Endothelium by Inhibiting Endothelial Lutheran/Basal Cell Adhesion Molecule (Lu/BCAM) through Phosphodiesterase 4A Activation

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α₄β₁ integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.     

Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co-implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular-derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).
Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.
Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE-cadherin and vascular endothelial growth factor-R2, and in their ability to endocytose acetylated low-density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro , as assessed by in vitro Matrigel sprouting assay. However in vivo , a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.
Conclusion:  Our results indicate that vascular-derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.     

Investigating native coronary artery endothelium in situ and in cell culture by scanning force microscopy

The purpose of our studies is to better understand the morphology and functioning of the arteries and their changes in pathogenesis. The most frequently used imaging techniques are intravascular ultrasound, magnetic resonance imaging, and optical coherence tomography. These methods do not image cell-level structural details and only provide biomechanical properties indirectly. We present a new protocol for imaging the endothelial surface and measuring elastic properties of vascular tissue by scanning force microscopy. Full-thickness sections of native pig coronary arteries were prepared. In addition, cultured human umbilical vein endothelial cells were studied as an in vitro model system and for comparison. We encountered a variety of difficulties mostly due to the softness of vascular tissue which required significant adaptations of standard equipment: (i) a new specimen holder designed to stably immobilize the coronary arteries; (ii) a phase-contrast microscope incorporated for assessing the status of the cultured endothelial cells and positioning the scanning force microscope (SFM) tip at a site of interest; and (iii) a continuous exchange of the culture medium at 37 degrees C to assure viability of the cells in the SFM over extended times. We were thus able to investigate both fresh arterial tissue and living endothelial cells in a near-physiological environment. We present initial SFM images of vascular tissue at a spatial resolution similar to scanning electron microscopy, but which also provide a closer view of the bona fide structure of native tissue. Novel morphological features such as distinct granular particles were observed. Moreover, we report initial measurements of vascular tissue surface stiffness, obtained by indentation-type SFM.     

Differential involvement of the hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis.

Hyaluronan (HA), an important glycosaminoglycan constituent of the extracellular matrix, has been implicated in angiogenesis. It appears to exert its biological effects through binding interactions with at least two cell surface receptors: CD44 and receptor for HA-mediated motility (RHAMM). Recent in vitro studies have suggested potential roles for these two molecules in various aspects of endothelial function. However, the relative contribution of each receptor to endothelial functions critical to angiogenesis and their roles in vivo have not been established. We therefore investigated the endothelial expression of these proteins and determined the effects of antibodies against RHAMM and CD44 on endothelial cell (EC) function and in vivo angiogenesis. Both receptors were detected on vascular endothelium in situ, and on the surface of cultured EC. Further studies with active blocking antibodies revealed that anti-CD44 but not anti-RHAMM antibody inhibited EC adhesion to HA and EC proliferation, whereas anti-RHAMM but not CD44 antibody blocked EC migration through the basement membrane substrate, Matrigel. Although antibodies against both receptor inhibited in vitro endothelial tube formation, only the anti-RHAMM antibody blocked basic fibroblast growth factor-induced neovascularization in mice. These data suggest that RHAMM and CD44, through interactions with their ligands, are both important to processes required for the formation of new blood vessels.     

Venous and arterial endothelial proteomics: mining for markers and mechanisms of endothelial diversity

Endothelial cells (ECs) line the inside of arterial and venous blood vessels in a continuous monolayer and have the important function of responding to environmental cues to regulate vascular tone and new blood vessel formation. They also have well-defined roles in supporting tumorigenesis, and alterations in their function lead to cardiovascular disease. Consequently, ECs have been studied extensively as a cellular model of both normal and abnormal physiology. Despite their importance and the increased utility of proteomic tools in medical research, there are relatively few publications on the topic of vascular endothelial proteomics. A thorough search of the literature mined 52 publications focused exclusively on arterial and/or venous endothelial proteomics. These studies mostly relied upon examination of whole-cell lysates from cultured human umbilical vein ECs to investigate in vitro effects of various molecules, such as VEGF in the context of altering human umbilical vein EC functions related to angiogenesis. Only a few of these publications focused solely on a proteomic characterization of ECs and our analysis further revealed a lack of published studies incorporating proteomic analysis of freshly isolated ECs from tissues or in vitro conditions that mimic in vivo variables, such as oxygen tension and shear stress. It is the purpose of this article to account for the diversity of vascular EC proteomic investigations and comment on the issues that have been and should be addressed in future work.