Article

An acellular matrix-bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model

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  • Pathologists Bio-Medical Labs
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Abstract

Circulating progenitor cells home to and engraft to sites of ischemia, mediated in part by the adhesion molecule L-selectin; however, accumulation in tissues such as the heart is low. In this study, an acellular collagen-based matrix containing sialyl Lewis(X) (sLe(X)), which binds L-selectin, was developed in order to enhance the endogenous progenitor cell therapeutic response. Its effect on progenitor cells and angiogenesis were assessed in vitro and using a hindlimb ischemia model with rats. In culture, the sLe(X)-collagen matrix recruited more CD133(+)CD34(+)L-selectin(+) cells than collagen-only matrix, with adhesion mediated by L-selectin binding. Increased angiogenic/chemotactic cytokine production and improved resistance to apoptosis appeared in cells cultured on sLe(X)-collagen matrix. In vivo, mobilization of endogenous circulating progenitor cells was increased, and greater recruitment of these and systemically injected human peripheral blood CXCR4(+)L-selectin(+) cells to sLe(X)-collagen treated limbs was observed compared to collagen-only. This condition was associated with differences in angiogenic/chemotactic cytokine levels, with greater arteriole density and increased perfusion in sLe(X)-collagen treated hindlimbs. With these factors taken together, we demonstrated that an acellular matrix-bound ligand approach can enhance the mobilization, recruitment, and therapeutic effects of endogenous and/or transplanted progenitor cells, possibly through paracrine and antiapoptotic mechanisms, and could be used to improve cell-based regenerative therapies.

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... However, the stem cell recruitment response is short-lived and tissue accumulation is low (Wojakowski et al., 2004; Fazel et al., 2006). Recruited CPCs are observed to be pro-angiogenic (Park et al., Suuronen et al., 2009) and are thought to augment functional recovery by promoting neovascularisation. This study aims to use the SDF-1 signalling mechanism in an effort to amplify the endogenous response to ischaemia and the recruitment of vasculogenic progenitor cells. ...
... As described previously (Suuronen et al., 2009), a collagen matrix was created on ice, using a crosslinking mixture containing a 1:1 molar ratio of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS; 13 mM) in 2(-N-morpholino) ethanesulfonic acid (MES) buffer, a solution of 1 % porcine type I atelocollagen (w/v; Nippon Ham, Tskuba, Japan) and 40 % chondroitin sulphate-C (CSC) (w/v; Wako Chemicals, Osaka, Japan). The cross-linked collagen solution was diluted with PBS before adjusting the pH to 7.2 ±0.2 using 1 N NaOH or HCl. ...
... CXCR4 is SDF-1's exclusive receptor and CXCR4 + CPCs are reduced over time, mechanisms other than SDF-1 release are needed to explain the increase in mobilised CPCs expressing fl k-1 and c-kit. We have previously shown that systemic transplantation of CPCs can induce a potent response from the host's CPCs (Suuronen et al., 2009). Additionally, this effect has been documented in humans, and CPC persistence in the circulation has been observed up to 1 year after cell transplantation (Turan et al., 2010). ...
Article
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Although many regenerative cell therapies are being developed to replace or regenerate ischaemic muscle, the lack of vasculature and poor persistence of the therapeutic cells represent major limiting factors to successful tissue restoration. In response to ischaemia, stromal cell-derived factor-1 (SDF-1) is up-regulated by the affected tissue to stimulate stem cell-mediated regenerative responses. Therefore, we encapsulated SDF-1 into alginate microspheres and further incorporated these into an injectable collagen-based matrix in order to improve local delivery. Microsphere-matrix impregnation reduced the time for matrix thermogelation, and also increased the viscosity reached. This double-incorporation prolonged the release of SDF-1, which maintained adhesive and migratory bioactivity, attributed to chemotaxis in response to SDF-1. In vivo, treatment of ischaemic hindlimb muscle with microsphere-matrix led to increased mobilisation of bone marrow-derived progenitor cells, and also improved recruitment of angiogenic cells expressing the SDF-1 receptor (CXCR4) from bone marrow and local tissues. Both matrix and SDF-1-releasing matrix were successful at restoring perfusion, but SDF-1 treatment appeared to play an earlier role, as evidenced by arterioles that are phenotypically older and by increased angiogenic cytokine production, stimulating the generation of a qualitative microenvironment for a rapid and therefore more efficient regeneration. These results support the release of implanted SDF-1 as a promising method for enhancing progenitor cell responses and restoring perfusion to ischaemic tissues via neovascularisation.
... In 1997, Asahara and colleagues (Asahara et al., 1997) identified bone marrow-derived endothelial progenitor cells (EPCs) in the circulating peripheral blood of adults that are capable of attaining EC characteristics in vitro. In further studies, it was demonstrated that these cells are able to repair injured blood vessels and to regenerate the function of ischaemic organs by vasculogenesis and angiogenesis (Hristov et al., 2003;Szmitko et al., 2006). The presence of these precious endothelial progenitor cells in adults offers scientists the possibility to generate an autologous endothelium on vascular grafts or stents (Szmitko et al., 2006) without the need of a vessel biopsy for isolation of ECs. ...
... In further studies, it was demonstrated that these cells are able to repair injured blood vessels and to regenerate the function of ischaemic organs by vasculogenesis and angiogenesis (Hristov et al., 2003;Szmitko et al., 2006). The presence of these precious endothelial progenitor cells in adults offers scientists the possibility to generate an autologous endothelium on vascular grafts or stents (Szmitko et al., 2006) without the need of a vessel biopsy for isolation of ECs. However, since the discovery of EPCs, there is a controversy about the identity of true circulating EPCs (Hur et al., 2004;Prater et al., 2007;Yoder et al., 2007). ...
... Research groups use the term "EPC" for a heterogeneous group of cells, such as for either "early" EPCs or "late" EPCs obtained in vitro. Other groups apply flow cytometry for quantification of EPCs, which are positive for CD34/CD133/VEGFR2 (Massa et al., 2005;Peichev et al., 2000), CD34/CD133 (Allanore et al., 2007), or CD34/VEGFR2 (Su et al., 2010). In turn others isolate CD34 positive cells using MACS (Magnetic Activated Cell Sorting) (Weber et al., 2004) and designate these cells as EPCs. ...
Article
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Circulating endothelial progenitor cells (EPCs) in the peripheral blood of adults represent an auspicious cell source for tissue engineering of an autologous endothelium on blood-contacting implants. Novel materials biofunctionalised with EPC-specific capture molecules represent an intriguing strategy for induction of selective homing of progenitor cells. The trapped EPCs can differentiate into endothelial cells and generate a non-thrombogenic surface on artificial materials. However, the success of this process mainly depends on the use of optimised capture molecules with a high selectivity and affinity. In recent years, various biomedical engineering strategies have emerged for in situ immobilisation of patient's own stem cells on blood contacting materials. The realisation of this in vivo tissue engineering concept and generation of an endothelium on artificial surfaces could exceedingly enhance the performance of not only small calibre vascular grafts and stents, but also, in general all blood-contacting medical devices, such as heart valves, artificial lungs, hearts, kidneys, and ventricular assist devices.
... Collectively, these findings suggest that a collagen-based material that can promote blood vessel growth may be an ideal candidate treatment for promoting skeletal muscle regeneration. We previously reported on a type I collagen-derived matrix that contained the oligosaccharide sialyl Lewis X (sLe X ), a ligand for the receptor L-selectin (Suuronen et al., 2009). L-selectin, a receptor for sLe X , is expressed on the surface membrane of circulating angiogenic cells (CACs), and has a role in regulating their homing and adhesion (Biancone et al., 2004). ...
... L-selectin, a receptor for sLe X , is expressed on the surface membrane of circulating angiogenic cells (CACs), and has a role in regulating their homing and adhesion (Biancone et al., 2004). When applied in a rat model of muscle ischaemia, the sLe X -matrix increased the number of c-kit + , CXCR4 + and VEGFR2 + CACs, augmented blood vessel regeneration and restored perfusion, while not eliciting a foreign body or harmful immune response (Suuronen et al., 2009). Given the importance of vasculature and the extracellular matrix in muscle regeneration, and since the sLe X -matrix can promote neovascularisation and its composition is based on type I collagen, we hypothesised that it may possess the ability to stimulate regenerative myogenesis. ...
... As previously described (Suuronen et al., 2009), 1 mM sLe X (Cedarlane Laboratories, Hornby, Canada) was prepared in 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer, at pH 5.0, containing 1:1 (molar equivalent) crosslinking mixture of N-ethyl-N-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS; 13 mM). This mixture was subsequently mixed on ice with 1 % porcine type I atelocollagen (w/v; Nippon Ham, Tskuba, Japan) with 40 % (w/v) chrondroitin sulphate-C (CS-C; Wako Chemicals, Osaka, Japan) and thoroughly mixed, then diluted with phosphate buffered saline (PBS). ...
Article
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Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.
... Given the biological (and possibly ethical) issues associated with cell transplantation, the prospect of using acellular biomaterials alone, including collagen-based matrices, for cardiac repair is also being explored (Wall et al. 2006 ;Badylak et al. 2009 ;Suuronen et al. 2009 ;Kuraitis et al. 2012a , b ;Johnson and Christman 2013 ). We have shown that incorporation of the oligosaccharide sialyl Lewis X (sLe X ), a ligand for the adhesion receptor L-selectin, into our collagen matrix enhanced the recruitment and engraftment of bone marrow-derived progenitor cells to ischemic hind limbs in rats. ...
... CXCR4 + cells ( arrowheads ) were often found to colocalize with the vasculature ( b and d ). Scale bars = 150 μm ( a and c ); 25 μm ( b and d )(Suuronen et al. 2009 ). Used with permission from: University of Ottawa Heart Institute, ( http://www.ottawaheart. ...
Chapter
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Cardiac disease is one of the most common causes of death in the world. Currently, surgically invasive therapies, such as implantable devices and, ultimately, heart transplantation, are the only remedies for end-stage congestive heart disease, which constitutes the final common pathway for all cardiac disorders. However, these surgical treatments are all mechanical and do not repair the damaged heart tissue. On the other hand, regenerative therapy promises to revolutionize the treatment of these patients and provides a biological and natural solution for cardiac repair. For example, biomaterials for the delivery of cells, growth factors and/or signaling molecules have been developed to treat the diseased heart. Biomaterials can provide suitable microenvironments to promote angiogenesis, deliver several key signals needed for repair processes, enhance engraftment and differentiation of cells, and modify cell function in different ways. Biomaterials can mimic the natural extracellular matrix, which supports the structure and functions of cells. Over the last decade, many biomaterials have been developed and used. In this chapter, we provide an overview of the biomaterials most commonly used for cardiac tissue engineering in preclinical studies and discuss their roles in cardiac repair processes.
... Others have focused on a less intervening strategy of using an implantable material that mediates and augments the body's natural responses. For example, the use of a Type l collagen-derived matrix that contained the oligosaccharide sialyl Lewis X (sLe X ), the natural ligand for the receptor L-Selectin, has been reported [249]. L-selectin is expressed on the membrane surface of endothelial progenitor cells (EPCs) and has a role in regulating their homing and adhesion to the ischemic areas [250]. ...
... L-selectin is expressed on the membrane surface of endothelial progenitor cells (EPCs) and has a role in regulating their homing and adhesion to the ischemic areas [250]. When used in a rat model of muscle ischemia, sLe X augmented the generation of new vasculature and restored perfusion [249]. This is of importance since revascularization to regain sufficient blood supply represents a major limitation for a successful therapeutic approach to enhance muscle regeneration after ischemia [251]. ...
... 2.1. Synthesis of collagen matrix Similar to methods described previously (Deng et al., 2010;Suuronen et al., 2009), 1% w/v type I porcine collagen (70% v/v total matrix; Nippon Ham) and 40% w/v chondroitin sulphate-C (CS-C; 3.4% v/v total matrix; Wako Chemicals) were mixed in 0.5 M 2-(N-morpholino) ethanesulphonic acid (MES) buffer (pH~6.0; EMD Chemicals). ...
... Collagen-and chitosan-based hydrogels are strong candidates for developing vascularized insulin-producing tissue for the treatment of type 1 diabetes. We have shown that these biomaterials can improve the retention, survival and function of transplanted and/or recruited cells (Suuronen et al., 2006(Suuronen et al., , 2009) because of their structure and compositional similarities to the ECM. The current study established a precedent for the feasibility in developing collagen-chitosan matrices ± laminin as ectopic islet transplant sites, capable of supporting islets and revascularization. ...
Article
Islet transplantation is an emerging strategy for treating patients with type 1 diabetes mellitus. Although the proof of concept for cellular replacement therapy in diabetes has been firmly established, vascularity of the transplant site and the long-term survival and function of transplanted islets remains suboptimal. In the present study, human circulating angiogenic cells (CACs) and porcine islet cells embedded in collagen-chitosan hydrogels, with and without laminin, were investigated as potential engineered biomaterials for the treatment of type 1 diabetes. Hydrogels were evaluated in vitro for their physical properties (compression, degradation, porosity and wettability) and cell compatibility. Increasing the chitosan content in the collagen-based hydrogel resulted in increased stiffness (p ≤ 0.04) and time to gelation (p < 0.001), but reduced porosity (from 22-28% to 16-19%). The material design formulations (10:1 vs 20:1 collagen:chitosan ratio) directly affected the cell properties. The viability of both human CACs and porcine islets embedded in the 20:1 collagen-chitosan matrix was higher at 24 h compared to the 10:1 formulation. For islet function, glucose stimulation indices for the 20:1 formulation at 24 h compared favourably with values reported in the literature, more so than the 10:1 formulations. While laminin improved the short-term viability of CACs, its presence did not confer any benefit to islet viability or function. Overall, the design features outlined in this study provided the degree of control required to establish viable tissue with potential for islet transplantation and neovascularization. Copyright © 2013 John Wiley & Sons, Ltd.
... Sialyl Lexis x has a high affinity for L-selectin present on circulating EPCs and has been shown to enhance mobilization and recruitment of EPCs. 92 Enhanced adhesion of EPCs utilizing was demonstrated utilizing a Sialyl Lexis xcollagen matrix in vitro and in a rat model. 92 More recently, hyaluronic acid (HA) oligosaccharide, a glycosaminoglycan with angiogenic and antithrombogenic properties, chains of varying lengths were grafted to PU-based films. ...
... 92 Enhanced adhesion of EPCs utilizing was demonstrated utilizing a Sialyl Lexis xcollagen matrix in vitro and in a rat model. 92 More recently, hyaluronic acid (HA) oligosaccharide, a glycosaminoglycan with angiogenic and antithrombogenic properties, chains of varying lengths were grafted to PU-based films. 93 While all three different lengths of HA proved more effective at limiting platelet adhesion and protein adsorption than PEG-or heparin-modified PU films, it was found that endothelial growth on the films was dependent on the molecular weight of the HA chains. ...
Article
Due to the lack of success in small-diameter (<6mm) prosthetic vascular grafts, a variety of strategies have evolved utilizing a tissue engineering approach. Much of this work has focused on enhancing the endothelialization of these grafts. A healthy, confluent endothelial layer provides dynamic control over hemodynamics, influencing and preventing thrombosis and smooth-muscle cell proliferation that can lead to intimal hyperplasia. Strategies to improve endothelialization of biodegradable polymeric grafts have encompassed both chemical and physical modifications to graft surfaces, many focusing on the recruitment of endothelial and endothelial progenitor cells. This review aims to provide a compilation of current and developing strategies that utilize in situ endothelialization to improve vascular graft outcomes, providing a context for the future directions of vascular tissue engineering strategies that do not require preprocedural cell seeding.
... Within the same primitive cell homing model we confirmed the presence of cells expressing the relevant receptors, CXCR4 and VLA-4. Interestingly, the SDF-1a mediated axis was activated in a very similar approach where host stem/progenitor cells were recruited by an activated collagen graft resulting in increased vascularization of the hind limb [25]. ...
Article
Optimizing current heart valve replacement strategies by creating living prostheses is a necessity to alleviate complications with current bioprosthetic devices such as calcification and degeneration. Regenerative medicine, mostly in vitro tissue engineering, is the forerunner of this optimization search, yet here we show the functionality of an in vivo alternative making use of 2 homing axes for stem cells. In rats we studied the signaling pathways of stem cells on implanted bioprosthetic tissue (photooxidized bovine pericardium (POP)), by gene and protein expression analysis. We found that SDF-1alpha/CXCR4 and FN/VLA4 homing axes play a role. When we implanted vascular grafts impregnated with SDF-1alpha and/or FN as carotid artery interpositions, primitive cells were attracted from the circulation. Next, bioprosthetic heart valves, constructed from POP impregnated with SDF-1alpha and/or FN, were implanted in pulmonary position. As shown by CD90, CD34 and CD117 immunofluorescent staining they became completely recellularized after 5 months, had a normal function and biomechanical properties and specifically the combination of SDF-1alpha and FN had an optimal valve-cell phenotype.
... L-selectin, an adhesion receptor found on leukocytes can also be found on EPCs [101]. In one study, the oligosaccharide sialyl Lexis x , which has a high affinity for L-selectin, was immobilized onto a collagen matrix and tested in vitro and in a murine model [102]. Enhanced endothelialisation properties were observed. ...
Article
Full-text available
The patency of synthetic cardiovascular grafts in the long run is synonymous with their ability to inhibit the processes of intimal hyperplasia, thrombosis and calcification. In the human body, the endothelium of blood vessels exhibits characteristics that inhibit such processes. As such it is not surprising that research in tissue engineering is directed towards replicating the functionality of the natural endothelium in cardiovascular grafts. This can be done either by seeding the endothelium within the lumen of the grafts prior to implantation or by designing the graft such that in situ endothelialisation takes place after implantation. Due to certain difficulties identified with in vitro endothelialisation, in situ endothelialisation, which will be the focus of this article, has garnered interest in the last years. To promote in situ endothelialisation, the following aspects can be taken into account: (1) Endothelial progenital cell mobilization, adhesion and proliferation; (2) Regulating differentiation of progenitor cells to mature endothelium; (3) Preventing thrombogenesis and inflammation during endothelialisation. This article aims to review and compile recent developments to promote the in situ endothelialisation of cardiovascular grafts and subsequently improve their patency, which can also have widespread implications in the field of tissue engineering.
... Wound healing is a complex process that requires a wellorchestrated interplay between different tissue structures and a large number of resident and infiltrating cell types. Especially, angiogenesis is an essential step in successful wound healing [1]. Progenitor cells take part in this orchestration, as they are able to regulate neovascularization and enhance healing by cytokine production [2]. ...
Article
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Introduction Stem cell transplantation is one of the most promising strategies to improve healing in chronic wounds as systemic administration of endothelial progenitor cells (EPC) enhances healing by promoting neovascularization and homing though a high amount of cells is needed. In the following study, we analysed whether local application can reduce the number of EPC needed achieving the same beneficial effect on wound healing. Material and Methods Wound healing after local or systemic treatment with EPC was monitored in vivo by creating standardized wounds on the dorsum of hairless mice measuring wound closure every second day. Systemic group received 2 × 10 ⁶ EPC i.v. and locally treated group 2 × 10 ⁵ EPC, locally injected. As control PBS injection was performed the same way. Expression of CD31, VEGF, CD90 and, SDF-1α was analysed immunohistochemically for evaluation of neovascularisation and amelioration of homing. Results Local (7.1 ± 0.45 SD) as well as systemic (6.1 ± 0.23 SD) EPC transplantation led to a significant acceleration of wound closure compared to controls (PBS local: 9.7 ± 0.5 SD, PBS systemic 10.9 ± 0.38 SD). Systemic application enhanced CD31 expression on day 6 after wounding and local EPC on 6 and 9 in comparison to control. VEGF expression was not significantly affected. Systemic and local EPC treatment resulted in a significantly enhanced SDF-1α and CD90 expression on all days investigated. Conclusion Local as well as systemic EPC treatment enhances wound healing. Moreover, beneficial effects are obtained with a tenfold decrease number of EPC when applied locally. Thus, local EPC treatment might be more convenient way to enhance wound healing as number of progenitor cells is limited.
... With ED-1 as a marker of macrophages, we assessed the presence of macrophages by IHC with an antibody specific for ED-1 (Fig. 3A). This antibody has been used by others [43], [44], [45]. The numbers of glomerular and tubulointerstitial macrophages were significantly higher in STZ vs. control (9.4±0.9 vs. 2±0.4 ...
Article
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... Numerous studies have shown that early cell losses from anoikis or inflammation, nonretention, lack of transdifferentiation, and nonviability in host tissues continue to limit exogenous cardiac cell therapy [3]. In contrast, data from our group and others have shown that the use of stand-alone biomatrices to promote endogenous repair may in itself lead to cardiovascular regeneration [4,5]. ...
Article
The mechanisms involved in myocardial regeneration and cardiac remodeling were examined by injecting porcine-derived small intestine submucosal extracellular matrix (SIS-ECM), with and without circulating angiogenic cells (CACs), in a mouse model of acute myocardial infarction (MI). Nine- to 10-week-old female C57BL/6J mice had the left anterior descending (LAD) coronary artery ligated. Seven days after ligation, 38 randomly allocated animals received echocardiographically guided intramyocardial injections of phosphate buffered saline (PBS), CACs, SIS-ECM, or SIS-ECM + CACs. Repeated echocardiography and immunohistochemical analysis were performed at 28 days after ligation. Baseline postligation left ventricular ejection fraction (LVEF) was equivalent in all groups. Twenty-one days after treatment, ejection fraction improved in the SIS-ECM + CAC treatment group (by 38% ± 2.12%) and the SIS-ECM treatment group (by 36% ± 3.71%), compared with the CAC-alone and PBS treatment groups (p < 0.1). Masson's trichrome staining showed reduced infarct size in SIS-ECM + CACs (34.2% ± 3.1%) and SIS-ECM alone (34.5% ± 4.7%) compared with CACs alone (47.3% ± 6.0%) and PBS (61.9% ± 5.5%; p < 0.002). Arteriolar density in periinfarct regions was enhanced in both SIS-ECM-treated groups (by ≥ 78% ± 7%; p = 0.03). More GATA4- and β-catenin-positive cardiac cells were found in the myocardium of SIS-ECM-treated animals. Intramyocardial delivery of SIS-ECM 7 days after MI in a mouse model reduced infarct size and improved myocardial vessel density and function; when combined with CACs it helped restore myocardial cellularity, suggesting a potential therapeutic role for SIS-ECM in cardiac regeneration.
... This combination has also been successful in cell delivery and implantation within target ischemic tissue using a collagen I/CS tissue-engineered matrix [427]. Other such biomaterials with well-defined chemical, topographical, and mechanical cues and even gradients of these physicochemical cues may also enhance endogenous progenitor cell homing and engrafting to sites of ischemia [428] and may serve as novel substrates for human circulating angiogenic cells to augment angiogenesis for the revascularization of ischemic and infarcted tissue [290]. However, native GAGs derived from human tissue are heterogeneous and structurally complex, and specific GAG moieties have been demonstrated to trigger specific cellular responses during cell division, motility and migration. ...
Article
Biomaterials have played an increasingly prominent role in the success of biomedical devices and in the development of tissue engineering, which seeks to unlock the regenerative potential innate to human tissues/organs in a state of deterioration and to restore or reestablish normal bodily function. Advances in our understanding of regenerative biomaterials and their roles in new tissue formation can potentially open a new frontier in the fast-growing field of regenerative medicine. Taking inspiration from the role and multi-component construction of native extracellular matrices (ECMs) for cell accommodation, the synthetic biomaterials produced today routinely incorporate biologically active components to define an artificial in vivo milieu with complex and dynamic interactions that foster and regulate stem cells, similar to the events occurring in a natural cellular microenvironment. The range and degree of biomaterial sophistication have also dramatically increased as more knowledge has accumulated through materials science, matrix biology and tissue engineering. However, achieving clinical translation and commercial success requires regenerative biomaterials to be not only efficacious and safe but also cost-effective and convenient for use and production. Utilizing biomaterials of human origin as building blocks for therapeutic purposes has provided a facilitated approach that closely mimics the critical aspects of natural tissue with regard to its physical and chemical properties for the orchestration of wound healing and tissue regeneration. In addition to directly using tissue transfers and transplants for repair, new applications of human-derived biomaterials are now focusing on the use of naturally occurring biomacromolecules, decellularized ECM scaffolds and autologous preparations rich in growth factors/non-expanded stem cells to either target acceleration/magnification of the body's own repair capacity or use nature's paradigms to create new tissues for restoration. In particular, there is increasing interest in separating ECMs into simplified functional domains and/or biopolymeric assemblies so that these components/constituents can be discretely exploited and manipulated for the production of bioscaffolds and new biomimetic biomaterials. Here, following an overview of tissue auto-/allo-transplantation, we discuss the recent trends and advances as well as the challenges and future directions in the evolution and application of human-derived biomaterials for reconstructive surgery and tissue engineering. In particular, we focus on an exploration of the structural, mechanical, biochemical and biological information present in native human tissue for bioengineering applications and to provide inspiration for the design of future biomaterials.
... 17 Similarly, greater vascular growth and presence of vWF þ endothelial cells was observed with the 1:1 and 5:1 collagenchitosan subcutaneous implants in our study. Further, recruited CXCR4 þ cells have been found to correlate with vascular density, 28,29 and recently, CXCR4 expression was found to determine the therapeutic potential of bonemarrow-derived mononuclear cells for neovascularization of ischemic tissue. 30 In this study, greater numbers of CXCR4 þ cells were found in collagen-chitosan implants, suggesting that increased recruitment of CXCR4 þ cells is a potential mechanism for the positive effect of chitosan materials on endothelial cell recruitment and blood vessel growth. ...
Article
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Cell therapy for the treatment of cardiovascular disease has been hindered by low cell engraftment, poor survival, and inadequate phenotype and function. In this study, we added chitosan to a previously developed injectable collagen matrix, with the aim of improving its properties for cell therapy and neovascularization. Different ratios of collagen and chitosan were mixed and chemically crosslinked to produce hydrogels. Swell and degradation assays showed that chitosan improved the stability of the collagen hydrogel. In culture, endothelial cells formed significantly more vascular-like structures on collagen–chitosan than collagen-only matrix. While the differentiation of circulating progenitor cells to CD31+ cells was equal on all matrices, vascular endothelial-cadherin expression was increased on the collagen–chitosan matrix, suggesting greater maturation of the endothelial cells. In addition, the collagen–chitosan matrix supported a significantly greater number of CD133+ progenitor cells than the collagen-only matrix. In vivo, subcutaneously implanted collagen–chitosan matrices stimulated greater vascular growth and recruited more von Willebrand factor (vWF+) and CXCR4+ endothelial/angiogenic cells than the collagen-only matrix. These results indicate that the addition of chitosan can improve the physical properties of collagen matrices, and enhance their ability to support endothelial cells and angiogenesis for use in cardiovascular tissue engineering applications.
... Complementary studies using a fabricated collagen matrix demonstrated reduced MSC relocation to other organs [32]. In a hindlimb ischemia model, combining collagen with sialyl Lewis(X), which binds L-selectin, resulted in increased recruitment of endothelial progenitor cells in vitro and increased mobilization of endogenous circulating precursor cells in vivo, together with a decrease in apoptosis [33]. Further augmentation of S/PC number was achieved by using engineered constructs as vehicles to deliver substances favoring their viability and/or survival. ...
Article
Full-text available
Inspired by studies demonstrating the potential for new myocyte formation within adult mammalian hearts, an ongoing explosion of research is elucidating the biology of cardiac myogenesis and angiogenesis. Multiple lines of research suggest that disease-associated activation of endogenous cardiac repair processes are often insufficient to overcome the cell death resulting from myocardial infarction and chronic heart failure. In this context, this review highlights current evidence supporting endogenous cardiac repair mechanisms in human hearts, recent progress with clinical application of myocardial cell therapy, and complementary efforts to manipulate endogenous myocardial repair processes using a variety of tissue engineering strategies. The goal of this overview is to demonstrate that the insights and opportunities derived from each of these lines of inquiry are mutually complementary for ultimately achieving the goal of therapeutic cardiac regeneration.
... 41 Interestingly, biomaterials such as an acellular collagen-based matrix containing sialyl Lewis(X), which binds L-selectin, increased mobilization, homing, and neovascularization of ischemic limbs through positive effects on endogenous and/or transplanted progenitor cells. 42 The process of rolling is reversible. Many progenitor cells or leukocytes that roll in vivo will not stop but dissociate from the vessel surface and reenter the bloodstream. ...
Article
Cell therapy has emerged as a promising option to treat myocardial infarction or heart failure; more than 1500 patients with cardiovascular diseases are treated with adult progenitor cells worldwide. The treatment of acute and chronic myocardial infarction with adult bone marrow-derived cells provided a modest benefit in most but not all studies. A number of plausible reasons have been discussed to explain the modest effects, sending researchers back to the bench to elucidate strategies to overcome the limitations of cell therapy and to develop more efficient approaches. Such strategies include the use of other sources to isolate adult progenitor cells (eg, adipose or cardiac tissue) or the generation of pluripotent cells by the reprogramming of somatic cell types. Successful cardiac cell therapy in clinical practice also depends on the efficient delivery and the appropriate integration and alignment of injected or infused cells. The pretreatment of cells with activators to augment cell homing and survival or the improvement of cell delivery tools may provide an opportunity to increase the number of active cells in ischemic or diseased tissues, thereby increasing their therapeutic potential. Finally, the identification of molecules guiding cardiac differentiation provides novel tools to enforce the formation of new functionally active cardiomyocytes to augment cardiac regeneration in its pure sense. This section provides a short overview of the different cell types that have been or are currently being tested for the treatment of patients with acute myocardial infarction or chronic heart failure (Figure 1). Most of the clinical studies performed so far have used bone marrow-derived mononuclear cells (BMCs) for treating patients with acute or chronic infarcts (for review, see elsewhere1). Overall, a modest but significant benefit was seen in meta-analyses of all published studies.2,3 Particularly in the acute setting, the infusion of BMCs via balloon catheters into …
... A reduction in Qdot concentration did not improve the labeling efficiency (Fig. 5A). Fluorescence microscopy demonstrated a dispersed staining pattern of the Qdots (at nM concentrations) within the matrix upon solidification (Fig. 5B), which is similar to another study that used EDC-NHS cross-linking to attach oligosaccharides to a collagen matrix (at mM concentration) [21]. FTIR analysis demonstrated the presence of stretch of the amide bonds, amide I and II, and eCOeNHe in the matrix (±Qdots) as reported in Table 1. ...
Article
Injectable biomaterials have shown promise for cardiac regeneration therapy. However, little is known regarding their retention and distribution upon application in vivo. Matrix imaging would be useful for evaluating these important properties. Herein, hexadecyl-4-[(18)F]fluorobenzoate ((18)F-HFB) and Qdot labeling was used to evaluate collagen matrix delivery in a mouse model of myocardial infarction (MI). At 1wk post-MI, mice received myocardial injections of (18)F-HFB- or Qdot-labeled matrix to assess its early retention and distribution (at 10min and 2h) by positron emission tomography (PET), or fluorescence imaging, respectively. PET imaging showed that the bolus of matrix at 10min redistributed evenly within the ischemic territory by 2h. Ex vivo biodistribution revealed myocardial matrix retention of ∼65%, which correlated with PET results, but may be an underestimate since (18)F-HFB matrix labeling efficiency was ∼82%. For covalently linked Qdots, labeling efficiency was ∼96%. Ex vivo Qdot quantification showed that ∼84% of the injected matrix was retained in the myocardium. Serial non-invasive PET imaging and validation by fluorescence imaging confirmed the effectiveness of the collagen matrix to be retained and redistributed within the infarcted myocardium. This study identifies matrix-targeted imaging as a promising modality for assessing the biodistribution of injectable biomaterials for application in the heart. Copyright © 2015 Elsevier Ltd. All rights reserved.
... Flow cytometry was performed on circulating GFP + cells collected by saphenous vein bleeds pre-operatively and at Days 1, 4, 7, and 14 post-surgery, as described previously. 20 Briefly, the mononuclear cell fraction was labelled with antibodies against the following antigens: c-kit (Southern Biotech, Birmingham, USA), CXCR4 (BD Biosciences, Mississauga, Canada), and flk-1 (mouse vascular endothelial growth factor receptor-2; eBioscience, San Diego, USA), and analysed with a FACSAria flow cytometer (BD Biosciences). The fold-change in the percentage of positive cells for early (Days 1 + 4) and late (Days 7 + 14) response times was calculated relative to baseline, as described previously. ...
Article
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Methylglyoxal accumulates in diabetes and impairs neovascularization. This study assessed whether over-expressing the methylglyoxal-metabolizing enzyme glyoxalase-1 (GLO1) in only bone marrow cells (BMCs) could restore neovascularization in ischemic tissue of streptozotocin-induced diabetic mice.Methods and ResultsAfter 24 h of hyperglycemic and hypoxic culture, BMCs from GLO1 over-expressing and wild-type (WT) diabetic mice were compared for migratory potential, viability and mRNA expression of anti-apoptotic genes (Bcl-2 and Bcl-XL). In vivo, BMCs from enhanced green fluorescent protein (GFP) mice that over-express GLO1 were used to reconstitute the BM of diabetic mice (GLO1-diabetics). Diabetic and non-diabetic recipients of WT GFP(+) BM served as controls (WT-diabetics and non-diabetics, respectively). Following hindlimb ischemia, the mobilization of BMCs was measured by flow cytometry. In hindlimbs, the presence of BM-derived angiogenic (GFP(+)CXCR4(+)) and endothelial (GFP(+)vWF(+)) cells, and arteriole density were determined by immunohistochemistry. Hindlimb perfusion was measured using Laser Doppler. GLO1-BMCs had superior migratory potential, increased viability, and greater Bcl-2 and Bcl-XL expression, compared to WT BMCs. In vivo, the mobilization of pro-angiogenic BMCs (CXCR4(+), c-kit(+) and Flk(+)) was enhanced post-ischemia in GLO1-diabetics compared to WT-diabetics. A greater number of GFP(+)CXCR4(+) and GFP(+)vWF(+) BMCs incorporated into hindlimb tissue of GLO1-diabetics and non-diabetics than in WT-diabetics. Arteriole and capillary density and perfusion were also greater in GLO1-diabetics and non-diabetics. This study demonstrates that protection from methylglyoxal uniquely in BM is sufficient to restore BMC function and neovascularization of ischemic tissue in diabetes and identifies GLO1 as a potential therapeutic target.
... Animals receiving FGF-2 treatment demonstrated higher levels of therapeutic cell-mobilizing cytokines G-CSF, MCP-1 and VEGF. Other studies have correlated enhanced angiogenesis in animal models with increased levels of G-CSF [30], MCP-1 [19,31] and VEGF [30]. Based on the serum cytokine profile observed, it may prove to be that another effect of our FGF-2 delivery system is to induce a systemic environment that is supportive of angiogenesis. ...
... The advanced strategy for EPC homing on a stent is coating capture molecules, such as antibodies [8][9][10], peptides [11,12], magnetic molecules [13,14], oligosaccharides [15,16], and aptamers [17][18][19][20] for fishing out EPCs directly from the bloodstream. However, only aptamer is selective for a single cell population [20,21]. ...
Article
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Restenosis and thrombosis are two major complications associated with vascular stents and grafts. The homing of circulating endothelial progenitor cells (EPCs) onto implant surfaces brings a new strategy to solve these problems by accelerating self -endothelialization in situ. Peptide aptamers with high affinity and specific recognition of EPCs can be immobilized to capture EPCs from the circulating blood. In this study, a biotinylated peptide aptamer (TPSLEQRTVYAK-GGGC-K-Biotin) for EPC, and bovine serum albumin (BSA) were co-immobilized onto titanium surface through avidin-biotin recognition to endow the surface with specific affinity for EPC and anti-platelet adhesion properties. Quartz crystal microbalance with dissipation (QCM-D), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and water contact angle measuring were adopted for coating characterization. EPC affinity and hemocompatibility of the coating were also investigated in vitro. The results demonstrated that aptamer and BSA co-immobilized surface significantly reduced platelet adhesion and fibrinogen adsorption/activation. Besides, such functional surface could remarkably enhance EPC adhesion, without affecting the behavior of endothelial cells (ECs) and smooth muscle cells (SMCs) obviously. The result shows the possibility of utilizing such a multifunctional surface in cardiovascular implants.
... Till date, the latest strategy for EPC homing on vascular stents has been coating the stents with capture molecules to mimic a natural homing substrate for fishing out EPCs directly from the bloodstream after implantation. Various types of capture molecules, including antibodies [11,12], peptides [13,14], oligosaccharides [15,16], and DNA-aptamers [17], have been utilized to attract EPCs on artificial devices. ...
Article
A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.
... Over the last decade, there are has been considerable research dedicated to the development of biomaterials, both synthetic and natural (see Table 1 for list of abbreviations), to aid in the healing process post-MI (Suuronen et al., 2006(Suuronen et al., , 2009Jourdan-Lesaux et al., 2010;Rane and Christman, 2011;Kuraitis et al., 2012;Ahmadi et al., 2014a). Biomaterials can take many forms from injectable hydrogels to solid patches and can serve a variety of purposes. ...
Article
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The healthy heart comprises many different cell types that work together to preserve optimal function. However, in a diseased heart the function of one or more cell types is compromised which can lead to many adverse events, one of which is myocardial infarction (MI). Immediately after MI, the cardiac environment is characterized by excessive cardiomyocyte death and inflammatory signals leading to the recruitment of macrophages to clear the debris. Proliferating fibroblasts then invade, and a collagenous scar is formed to prevent rupture. Better functional restoration of the heart is not achieved due to the limited regenerative capacity of cardiac tissue. To address this, biomaterial therapy is being investigated as an approach to improve regeneration in the infarcted heart, as they can possess the potential to control cell function in the infarct environment and limit the adverse compensatory changes that occur post-MI. Over the past decade, there has been considerable research into the development of biomaterials for cardiac regeneration post-MI; and various effects have been observed on different cell types depending on the biomaterial that is applied. Biomaterial treatment has been shown to enhance survival, improve function, promote proliferation, and guide the mobilization and recruitment of different cells in the post-MI heart. This review will provide a summary on the biomaterials developed to enhance cardiac regeneration and remodeling post-MI with a focus on how they control macrophages, cardiomyocytes, fibroblasts, and endothelial cells. A better understanding of how a biomaterial interacts with the different cell types in the heart may lead to the development of a more optimized biomaterial therapy for cardiac regeneration.
... In order to increase the number of circulating EPC, a strategy will need to be devised to mobilize the EPC from the bone marrow, since only very few EPC are thought to be circulating in peripheral blood. Several factors are known to increase the number of circulating EPC, including SDF-1, IL-8, Ang-1, and granulocyte colony-stimulating factor (G-CSF). [78][79][80][81][82] The main challenge lies in creating a signal that is strong enough to attract EPC and that is above the "background noise" associated with inflammation and tissue injury associated with biomaterial implantation. Moreover, since the implanted biomaterial will not be lined with EC, strategies to inhibit thrombosis and prevent undesirable protein adsorption and cell adhesion will be required. ...
... A central challenge in this context is the attraction, adhesion, and proliferation of endothelial progenitor cells (EPCs) or endothelial cells (ECs) to form a complete endothelium. Several strategies to address this issue have been described: immobilization of antibodies targeting markers for EPCs such as vascular endothelial growth factor receptor 2 (VEGFR2) and platelet endothelial cell adhesion molecule (PECAM-1) [14,15]; modification of the surface with peptides such as the Arg-Gly-Asp (RGD) or Cys-Ala-Gly (CAG) sequence [16,17]; immobilization of growth factors such as the vascular endothelial growth factor (VEGF) or stromal cell-derived factor-1 (SDF-1) [18,19]; immobilization of oligonucleotides and aptamers [20,21]; and surface modification with oligosaccharides and phospholipids [22,23]. However, it is necessary to develop surfaces with improved biocompatible, bioactive, targeted, and stable biofunctionalization [24]. ...
Article
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Appropriate mechanical properties and fast endothelialization of synthetic grafts are key to ensure long-term functionality of implants. We used a newly developed biostable polyurethane elastomer (TPCU) to engineer electrospun vascular scaffolds with promising mechanical properties (E-modulus: 4.8 ± 0.6 MPa, burst pressure: 3326 ± 78 mmHg), which were biofunctionalized with fibronectin (FN) and decorin (DCN). Neither uncoated nor biofunctionalized TPCU scaffolds induced major adverse immune responses except for minor signs of polymorph nuclear cell activation. The in vivo endothelial progenitor cell homing potential of the biofunctionalized scaffolds was simulated in vitro by attracting endothelial colony-forming cells (ECFCs). Although DCN coating did attract ECFCs in combination with FN (FN + DCN), DCN-coated TPCU scaffolds showed a cell-repellent effect in the absence of FN. In a tissue-engineering approach, the electrospun and biofunctionalized tubular grafts were cultured with primary-isolated vascular endothelial cells in a custom-made bioreactor under dynamic conditions with the aim to engineer an advanced therapy medicinal product. Both FN and FN + DCN functionalization supported the formation of a confluent and functional endothelial layer.
... SDF-1 α will bind with the EPC receptor CXCR4 while VEGF binds to VEGFR-2, which signifi cantly increases cellular adhesion (Shen et al. , 2008;Singh et al. , 2011;Yu et al. , 2012). Polysaccharides such as sialyl Lewis (X) are also being incorporated into biomaterials to promote both the recruitment and retention of EPCs through interaction with L -selectin receptors (Sanders et al. , 1996;Biancone et al. , 2004;Suuronen et al. , 2009;Kuraitis et al. , 2012). Immobilizing CD34 antibodies on hyaluronic acid-heparin hydrogels has also been successful in promoting EPC adhesion (Camci-Unal et al. , 2012). ...
Chapter
Endothelial progenitor cell (EPC) therapy has emerged as a promising treatment in cardiac regeneration. EPCs are capable of promoting neovascularization within ischemic tissues, but past studies have had limited success in vivo due to the poor retention and survival of cells within the injured area. In addition, the environment into which the cells are being transplanted may negatively influence the therapeutic potential of the transplanted cells. To overcome these hurdles, various biomaterials are being engineered to include a variety of proteins and peptides to increase cellular adhesion, survival and angiogenesis. This chapter will describe how such strategies are expected to enhance EPCs, and their ability to promote cardiac repair and regeneration.
... A central challenge in this context is the attraction, adhesion, and proliferation of endothelial progenitor cells (EPCs) or endothelial cells (ECs) to form a complete endothelium. Several strategies to address this issue have been described: immobilization of antibodies targeting markers for EPCs such as vascular endothelial growth factor receptor 2 (VEGFR2) and platelet endothelial cell adhesion molecule (PECAM-1) [14,15]; modification of the surface with peptides such as the Arg-Gly-Asp (RGD) or Cys-Ala-Gly (CAG) sequence [16,17]; immobilization of growth factors such as the vascular endothelial growth factor (VEGF) or stromal cell-derived factor-1 (SDF-1) [18,19]; immobilization of oligonucleotides and aptamers [20,21]; and surface modification with oligosaccharides and phospholipids [22,23]. However, it is necessary to develop surfaces with improved biocompatible, bioactive, targeted, and stable biofunctionalization [24]. ...
Article
Coronary artery disease is a leading cause of death in developed nations. As the disease progresses, myocardial infarction can occur leaving areas of dead tissue in the heart. To compensate, the body initiates its own repair/regenerative response in an attempt to restore function to the heart. These efforts serve as inspiration to researchers who attempt to capitalize on the natural regenerative processes to further augment repair. Thus far, researchers are exploiting these repair mechanisms in the functionalization of soft materials using a variety of growth factor-, ligand- and peptide-incorporating approaches. The goal of functionalizing soft materials is to best promote and direct the regenerative responses that are needed to restore the heart. This review summarizes the opportunities for the use of functionalized soft materials for cardiac repair and regeneration, and some of the different strategies being developed.
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Experimental studies in animals and recent human clinical trials have revealed the current limitations of cellular transplantation, which include poor cell survival, lack of cell engraftment, and poor differentiation. Evidence in animals suggests that use of a 3-dimensional scaffold may enhance cell therapy and engineer myocardial tissue by improving initial cell retention, survival, differentiation, and integration. Several scaffolds of synthetic or natural origin are under development. Until now, contractility has been demonstrated in vitro only in biological scaffolds prepared from decellularized organs or tissue, or in collagenic porous scaffold obtained by crosslinking collagen fibers. While contractility of a cellularized collagen construct is poor, it can be greatly enhanced by tumor basement membrane extract. Recent advances in biochemistry have shown improved cell-matrix interactions by coupling adhesion molecules to achieve an efficient and safe bioartificial myocardium with no tumoral component. Fixation of adhesion molecules may also be a way to enhance cell homing and/or differentiation to increase local angiogenesis. Whatever the clinically successful combination ultimately proves to be, it is likely that cell therapy will require providing a supportive biochemical, physical, and spatial environment that will allow the cells to optimally differentiate and integrate within the target myocardial tissue.
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Stem cell therapy for cardiac disease may be facing two major problems nowadays: although vasculogenesis likely occurs as a result of cell therapy, its clinical applications are limited and significant, integrated cardiomyogenesis has not demonstratively been shown to occur, even in the experimental setting, with any other source than embryonic or other pluripotent stem cells. In this article, we highlight several factors that will need to be optimized if we are to achieve clinically effective cardiomyogenesis, such as the identification of optimal stem cell populations, and the ideal time and methods for cell transplantation. So far, educated attempts at achieving transplanted stem cell-induced myogenesis have largely failed outside of the embryonic stem cell realm, and we present the rationale for also considering acellular techniques, which may enhance the potential of endogenous progenitor populations. In today's cardiovascular field, once a cardiomyocyte is lost it is lost for good, without any form of direct therapeutic option. For these reasons, cell therapy justifies our continued attention and efforts, and may constitute the holy grail of cardiovascular therapeutics.
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Cardiovascular disease continues to be a major cause of death in the Western world and has been extending into areas previously seemingly immune to its effects. Catheter-based interventions and coronary artery bypass surgery have markedly improved cardiovascular health, but a number of patients with coronary artery disease cannot undergo repeated interventions or they receive an incomplete revascularization with standard revascularization methods, which has been associated with a poor clinical outcome. Despite early demonstration of improvement in myocardial perfusion and function with growth factor, gene therapy or cellular therapies, clinical studies have found little if any real benefit. The discordance between positive pre-clinical studies and essentially negative clinical trials may in part be explained by a number of factors including abnormal vascular signaling, oxidative stress, a hostile local myocardial environment and technical issues related to the administration of these therapies. Patients with end-stage coronary disease are vastly different from the young, healthy animals that are generally used for pre-clinical testing. The presence of diabetes, hypercholesterolemia, and other conditions associated with endothelial dysfunction and coronary disease and altered vascular signaling can significantly limit the effectiveness of growth factors on the development of collateral vessels. This paper summarizes the results of regenerative therapies for the treatment of coronary disease and discusses the reasons why growth factor protein, gene therapies and cellular therapies have not been overall successful to date.
Article
Aim: To investigate the behavior of porcine-derived decellularized muscular matrix (DMM) in vitro and compare the performance of this biological mesh with that of acellular dermal matrix from pig in a full abdominal wall defect model. Materials & methods: To describe the in vitro properties of the DMM scaffold with extracellular matrix histological analysis, growth factor quantification and scanning electron microscopy analysis. To compare structural and functional remodeling between acellular dermal matrix and DMM implants in a rodent full abdominal wall defect model. Results & conclusion: The results demonstrated that cellular components were effectively removed in the DMM scaffold, which also maintained a 3D architecture, biochemical components and strong mechanical properties. In vivo experiments confirm that the DMM mesh could promote remodeling and reconstruction of functional skeletal muscle tissue.
Article
Biomaterials that have the ability to augment angiogenesis are highly sought-after for applications in regenerative medicine, particularly for revascularization of ischemic and infarcted tissue. We evaluated the culture of human circulating angiogenic cells (CAC) on collagen type I-based matrices, and compared this to traditional selective-adhesion cultures on fibronectin. Culture on a collagen matrix supported the proliferation of CD133(+) and CD34(+)CD133(+) CACs. When subjected to serum starvation, the matrix conferred a resistance to cell death for CD34(+) and CD133(+) progenitors and increased phosphorylation of Akt. After 4days of culture, phenotypically enriched populations of endothelial cells (CD31(+)CD144(+)) and progenitor cells (CD34(+)CD133(+)) emerged. Culture on matrix upregulated the phosphorylation and activation of ERK1/2 pathway members, and matrix-cultured cells also had an enhanced functional capacity for adhesion and invasion. These functional improvements were abrogated when cultured in the presence of ERK inhibitors. The formation of vessel-like structures in an angiogenesis assay was augmented with matrix-cultured cells, which were also more likely to physically associate with such structures compared to CACs taken from culture on fibronectin. In vivo, treatment with matrix-cultured cells increased the size and density of arterioles, and was superior at restoring perfusion in a mouse model of hindlimb ischemia, compared to fibronectin-cultured cell treatment. This work suggests that a collagen-based matrix, as a novel substrate for CAC culture, possesses the ability to enrich endothelial and angiogenic populations and lead to clinically relevant functional enhancements.
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Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in cell delivery for cardiac regeneration. While the materials' properties are often characterized, how these properties (and in particular gelation) are affected by the addition of the therapeutic agent(s) they are designed to deliver is often overlooked. The aim of this study was to examine the interactive effects between collagen-based hydrogels and different additives (cells and microspheres). The results demonstrated that the incorporation of either cells or microspheres to a collagen hydrogel decreased its gelation time and increased its viscosity. Increased concentrations of the EDC/NHS cross-linker resulted in greater loss of cell viability. However, it was found that this cell loss could be minimized by delivering cells with the cross-linker scavenger glycine. A better understanding of how materials and cells (and other additives) respond to each other will help towards the goal of improving scaffolds being developed for regenerative therapy.
Article
Introduction: The diseased host milieu, such as endothelial dysfunction (ED), decreased NO bioavailability, and ischemic/inflammatory post-MI environment, hamper the clinical success of existing cardiac regenerative therapies. Area covered: In this article, current strategies including pharmacological and nonpharmacological approaches for improving the diseased host milieu are reviewed. Specifically, the authors provide focus on: i) the mechanism of ED in patients with cardiovascular diseases, ii) the current results of ED improving strategies in pre-clinical and clinical studies, and iii) the use of biomaterials as a novel modulator in damaged post-MI environment. Expert opinion: Adjunct therapies which improve host endothelial function have demonstrated promising outcomes, potentially overcoming disappointing results of cell therapy in human studies. In the future, elucidation of the interactions between the host tissue and therapeutic agents, as well as downstream signaling pathways, will be the next challenges in enhancing regenerative therapy. More careful investigations are also required to establish these agents’ safety and efficacy for wide usage in humans.
Article
Myopathies of skeletal muscle are prevalent diseases worldwide. To address this, regenerative therapies are being developed to restore perfusion to ischemic muscle and to reverse muscle wasting. There are adult stem cell populations that inherently possess these therapeutic properties; however, cell transplantation trials in the clinic have shown modest results at best, being limited by poor cell persistence and viability post-transplantation, and by cell relocation to non-target sites. Many materials exist that can elicit and enhance beneficial cell responses - these materials can be applied directly, or used as stem cell delivery vehicles, for regenerative therapies. In particular, components of the body's extracellular matrices may be advantageous for therapeutic application because cells already have a pre-disposition for recognizing them, and also because their usage carries a low probability of inducing negative immune responses. This review will survey the major components of the extracellular matrix and their interactions with relevant stem cell populations for the regeneration of muscle. Future material-based therapies will benefit from a more precise control over therapeutic cell populations implicated in the regenerative response.
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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.
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Due to the very limited ability of cardiac tissue to self-regenerate, the replacement of damaged cardiomyocytes and the repair of damaged extracellular matrix (ECM) are highly sought-after therapeutic strategies. Cell transplantation in ECM scaffolds has been shown to improve retention, phenotype, and function in vascular and muscle repair. In addition to cellular patches that involve the use of biomaterial scaffolds in combination with cells, acellular patches may have a role in intrinsically recruiting cells to damaged areas. This review focuses on the clinically relevant ECM scaffolds, their interactions with cells to stimulate functions such as adhesion, migration, proliferation, and differentiation, and their intrinsic role in ECM remodeling leading to vascular and possibly myocardial repair.
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Cardiovascular disease (CVD) is a leading cause of death and hospitalization worldwide. The need for small caliber vessels ( < 6mm) to treat CVD patients has grown; however the availability of autologous vessels in cardiac and peripheral bypass candidates is limited. The search for an alternative vessel source is widespread with both natural and synthetic tissue engineered materials being investigated as scaffolds. Despite decades of exhaustive studies with decellularized extracellular matrices (ECM) and synthetic graft materials, the field remains in search of a commercially viable biomaterial construct substitute. While the previous materials have been assessed by evaluating their compatibility with fibroblasts, smooth muscle cells and endothelial cells, current materials are being conceived based on their interactions with stem cells, progenitor cells and monocytes, as the latter may hold the key to repair and regeneration. The graft's ability to recruit and maintain these cells has become a major research focus. The successful tissue engineering of a small caliber vessel graft requires the use of optimal material chemistry and biological function to promote cell recruitment into the graft while maintaining each functional phenotype during vessel tissue maturation. The discussion of these significant research challenges constitutes the focus of this review.
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Surface biofunctional modification of cardiovascular implants via the conjugation of biomolecules to prevent thrombosis and restenosis formation and to accelerate endothelialization has attracted considerable research interest. In this study, we aimed to develop a multifunctional surface that could exhibit good hemocompatibility and function well in inducing desirable vascular cell-material interactions. The multifunctional coating ([email protected]/* */), containing phosphorylcholine groups and endothelial progenitor cell (EPC)-specific peptides (PT), was prepared on titanium (Ti) surfaces via chemical conjugation. The results of platelet adhesion, activation, fibrinogen denaturation, and whole blood dynamic adhesion testing indicated that the [email protected]/* */ coating presented a better hemocompatibility when compared with bare Ti and other control samples. In vitro EPC and smooth muscle cell (SMC) cultures showed that the [email protected]/* */ coating significantly promoted the adhesion and proliferation of EPCs and inhibited the attachment and proliferation of SMCs. In vivo animal tests further confirmed that the [email protected]/* */ coating effectively inhibited thrombus formation and intimal hyperplasia while supporting endothelium regeneration. These results effectively suggest that the [email protected]/* */ coating may be promising as a coating on cardiovascular implants.
Article
Introduction: The feasibility and safety of bone marrow cell (BMC) therapy for cardiac repair following myocardial infarction has been demonstrated in clinical studies, albeit with relatively modest structural and functional benefits. In response to the shortcomings of BMC therapy, the use of biomaterials to enhance cell transplantation is being investigated. Areas covered: The authors first review what has been learned from BMC therapies for the treatment of myocardial infarction in animal models and in clinical trials. Some issues that may be limiting the efficacy of BMC therapy are then described. Lastly, they summarize several biomaterial approaches that have been reported to improve transplanted cell retention and functional outcome, and then focus on how a material can enhance cell function such as proliferation, viability, endothelial differentiation and angiogenic potential. Expert opinion: Improvements are needed if BMC therapy is to become a viable treatment in the clinic. There is optimism that a biomaterial strategy will lead to superior results compared to the cell therapy alone. Through the identification of underlying cell-biomaterial mechanisms, the establishment of comparative standards, and an awareness of the lessons learned from cell therapy trials, biomaterial-enhanced BMC therapy may become an option for the treatment of heart disease patients.
Article
Endothelial progenitor cell (EPC) capturing techniques have led to revolutionary strategies that can improve the performance of cardiovascular implant devices and engineered tissues by enhancing re-endothelialization and angiogenesis. However, these strategies are limited by controversies regarding the phenotypic identities of EPCs as well as their inability to target and prevent the other afflictions associated with current therapies, namely thrombosis and neointimal hyperplasia. Therefore, the goal of this study is to study the efficacy of a bioinspired multifunctional nanomatrix in recruiting and promoting the differentiation of EPCs towards an endothelial lineage. The bioinspired nanomatrix combines multiple components including self-assembled peptide amphiphiles (PAs) that include cell adhesive ligands, nitric oxide (NO) producing donors, and enzyme mediated degradable sequences to achieve an endothelium mimicking character. In this study, human peripheral blood mononuclear cells (PBMNCs) were isolated and cultured on the bioinspired multifunctional nanomatrix. Initial cell adhesion, lectin staining, Ac-LDL uptake, and expression of endothelial markers including CD31, CD34, vWF, and VEGFR2 were analyzed. The results from this study indicate that the NO releasing bioinspired multifunctional nanomatrix promotes initial adhesion of EPCs when compared to control surfaces. The expression of endothelial markers is also increased on the bioinspired multifunctional nanomatrix, suggesting that it directs the differentiation of EPCs towards an endothelial phenotype. The bioinspired nanomatrix therefore provides a novel biomaterial based platform for capturing as well as directing EPC behavior. Therefore this study has the potential to positively impact the patency of cardiovascular devices such as stents and vascular grafts as well as enhanced angiogenesis for ischemic or engineered tissues.
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Regeneration of myocardium through regenerative therapy and tissue engineering is appearing as a prospective treatment modality for patients with end-stage heart failure. Focusing on this area, this review highlights the new developments and challenges in the regeneration of myocardial tissue. The role of various cell sources, calcium ion and cytokine on the functional performance of regenerative therapy is discussed. The evolution of tissue engineering and the role of tissue matrix/scaffold, cell adhesion and vascularisation on tissue engineering of cardiac tissue implant are also discussed.
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Thrombogenicity of foreign surfaces is the major obstacle in cardiovascular interventions. Despite enormous advances in biomaterials research, the hemocompatibility of blood-contacting materials is still not satisfactory and the native endothelium still represents the ideal surface for blood contact. Circulating adult endothelial progenitor cells (EPCs) in the human blood provide an excellent source of autologous stem cells for the in vivo self-endothelialization of blood-contacting materials. For this purpose, material surfaces can be coated with capture molecules mimicking natural homing factors to attract circulating EPCs. Hitherto, several ligands, such as aptamers, monoclonal antibodies, peptides, selectins and their ligands, or magnetic molecules, are used to biofunctionalize surfaces for the capturing of EPCs directly from patient's bloodstream onto blood-contacting materials. Subsequently, attracted EPCs can differentiate into endothelial cells and generate an autologous endothelium. The in vivo self-endothelialization of blood-contacting materials prevents the recognition of them as a foreign body; this opens up revolutionary new prospects for future clinical stem-cell and tissue engineering strategies.
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Endothelial progenitor cell (EPC) -seeded intravascular stents are one of the primary potential strategies for treating cardiovascular diseases. Since their emergence, endovascular stents have undergone two phases of development: conventional bare-metal stents and drug-eluting stents. However, both types of stents are associated with the clinical problems of intimal hyperplasia and stenosis. At present, the use of slow-release stents that induce endothelial progenitor cell homing can prevent intimal stenosis and preserve vascular patency. Endothelial progenitor cell-seeded intravascular stents represent a future developmental direction for vascular engineering technology.
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Peripheral arterial disease remains an often devastating condition, particularly in patients with diabetes, because of the high rate of functional disability, amputation and death. For those patients for whom conventional endovascular or surgical revascularization procedures have been unsuccessful, new options are eagerly awaited, among which cell therapy has gained increasing interest. Most clinical trials of cell therapy have used multiple intramuscular injections of bone marrow-derived mononuclear cells that have yielded encouraging suggestions of efficacy. The prevailing opinion is that the benefits of cell therapy are not a result of the structural integration of grafted cells within new vessels, but of the paracrine activation of angiogenesis, arteriogenesis and vasculogenesis pathways by the cytokines, chemokines and growth factors released from such cells. An analysis of cell therapy clinical trial outcomes has also identified several key issues that need to be addressed, including the optimal cell type, source and dosing, the most effective route for cell transfer, and methods for enhancing survival of the cellular graft. Finally, because of the strong placebo effect that may confound interpretation of outcome measures, rigorously randomized controlled trials are mandatory in order to assess more thoroughly whether cell therapy will be beneficial for patients with peripheral arterial disease.
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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.-McNeill, B., Vulesevic, B., Ostojic, A., Ruel, M., Suuronen, E. J. Collagen matrix-induced expression of integrin αVß3 in circulating angiogenic cells can be targeted by matricellular protein CCN1 to enhance their function. © FASEB.
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Endothelial progenitor cells (EPCs) are adult stem cells that play a central role in neovascularization. EPCs are mobilized from bone marrow into peripheral blood, attach to existing endothelial cells, and then transmigrate across the endothelium into tissues, where they proliferate, differentiate, and form new blood vessels. In the process, EPCs are exposed to shear stress, a biomechanical force generated by flowing blood and tissue fluid flow. When cultured EPCs are exposed to controlled levels of shear stress in a flow-loading device, their bioactivities in terms of proliferation, anti-apoptosis, migration, production of bioactive substances, anti-thrombosis, and tube formation increase markedly. Expression of endothelial marker genes and proteins by EPCs also increases in response to shear stress, and they differentiate into mature endothelial cells. Great advances have been made in elucidating the mechanisms by which mature endothelial cells sense and respond to shear stress, but not in EPCs. Further study of EPC responses to shear stress will be necessary to better understand the physiological and pathophysiological roles of EPCs and to apply EPCs to new therapies in the field of regenerative medicine.
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The aim of this study was to investigate the ability of peptides and peptide combinations to support circulating endothelial colony forming cell (ECFC) rolling and adhesion under shear flow, informing biomaterial design in moving toward rapid cardiovascular device endothelialization. ECFCs have high proliferative capability and can differentiate into endothelial cells, making them a promising cell source for endothelialization. Both single peptides and peptide combinations designed to target integrins α4β1 and α5β1 were coupled to poly(ethylene glycol) hydrogels, and their performance was evaluated by monitoring velocity patterns during the ECFC rolling process, in addition to firm adhesion (capture). Tether percentage and velocity fluctuation, a parameter newly defined here, were found to be valuable in assessing cell rolling velocity patterns and when used in combination were able to predict cell capture. REDV-containing peptides binding integrin α4β1 have been previously shown to reduce ECFC rolling velocity but not to support firm adhesion. This study finds that the performance of REDV-containing peptides in facilitating ECFC dynamic adhesion and capture can be improved by combination with α5β1 integrin-binding peptides, which support ECFC static adhesion. Moreover, when similar in length, the peptide combinations may have synergistic effects in capturing ECFCs. With matching lengths, the peptide combinations including CRRETAWAC(cyclic)+REDV, P_RGDS+KSSP_REDV, and P_RGDS+P_REDV showed high values in both tether percentage and velocity fluctuation and improvement in ECFC capture compared to the single peptides at the shear rate of 20 s–1. These newly identified peptide combinations have the potential to be used as vascular device coatings to recruit ECFCs. Statement of Significance Restoration of functional endothelium following placement of stents and vascular grafts is critical for maintaining long-term patency. Endothelial colony forming cells (ECFCs) circulating in blood flow are a valuable cell source for rapid endothelialization. Here we identify and test novel peptides and peptide combinations that can potentially be used as coatings for vascular devices to support rolling and capture of ECFCs from flow. In addition to the widely used assessment of final ECFC adhesion, we also recorded the rolling process to quantitatively evaluate the interaction between ECFCs and the peptides, obtaining detailed performance of the peptides and gaining insight into effective capture molecule design. Peptide combinations targeting both integrin α4β1 and integrin α5β1 showed the highest percentages of ECFC capture.
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Circulating blood-derived vasculogenic cells improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. The question remains to what extent a physical incorporation is contributing to the beneficial effects of cell therapy. By using the inducible suicide gene thymidine kinase to deplete transplanted cells, we determined the contribution of physical incorporation in 3 animal models. After acute myocardial infarction, depletion of cells 14 days after infusion resulted in a reduction of capillary density and a substantial deterioration of heart function. Likewise, neovascularization of Matrigel plugs and ischemic limbs was significantly suppressed when infused cells were depleted 7 days after infusion. Induction of cell death in the previously transplanted cells reduced perfusion and led to vascular leakage as evidenced by Evans blue extravasation. These results indicate that physical incorporation and persistence of cells contribute to cell-mediated improvement of neovascularization and cardiac function. Long-term paracrine activities and/or cell intrinsic mechanisms may have contributed to the maintenance of functional improvement.
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Emerging data suggest that a subset of circulating human CD34(+) cells have phenotypic features of endothelial cells. Whether these cells are sloughed mature endothelial cells or functional circulating endothelial precursors (CEPs) is not known. Using monoclonal antibodies (MoAbs) to the extracellular domain of the human vascular endothelial receptor-2 (VEGFR-2), we have shown that 1.2 +/- 0.3% of CD34(+) cells isolated from fetal liver (FL), 2 +/- 0.5% from mobilized peripheral blood, and 1.4 +/- 0.5% from cord blood were VEGFR-2(+). In addition, most CD34(+)VEGFR-2(+) cells express hematopoietic stem cell marker AC133. Because mature endothelial cells do not express AC133, coexpression of VEGFR-2 and AC133 on CD34(+) cells phenotypically identifies a unique population of CEPs. CD34(+)VEGFR-2(+) cells express endothelial-specific markers, including VE-cadherin and E-selectin. Also, virtually all CD34(+)VEGFR-2(+) cells express the chemokine receptor CXCR4 and migrate in response to stromal-derived factor (SDF)-1 or VEGF. To quantitate the plating efficiency of CD34(+) cells that give rise to endothelial colonies, CD34(+) cells derived from FL were incubated with VEGF and fibroblast growth factor (FGF)-2. Subsequent isolation and plating of nonadherent FL-derived VEGFR-2(+) cells with VEGF and FGF-2 resulted in differentiation of AC133(+ )VEGFR-2(+) cells into adherent AC133(-)VEGFR-2(+)Ac-LDL(+ )(acetylated low-density lipoprotein) colonies (plating efficiency of 3%). In an in vivo human model, we have found that the neo-intima formed on the surface of left ventricular assist devices is colonized with AC133(+)VEGFR-2(+) cells. These data suggest that circulating CD34(+) cells expressing VEGFR-2 and AC133 constitute a phenotypically and functionally distinct population of circulating endothelial cells that may play a role in neo-angiogenesis.
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Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS(R) multisort method. Clones of MDSCs, which were Sca-1+/CD34-/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.
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The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.
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Cell implantation into areas of myocardial infarction (cellular cardiomyoplasty) may be limited in efficacy because of the lack of blood supply to these areas of myocardium, resulting in early loss of transplanted cells. We therefore tested the hypothesis that pretreatment of infarcted myocardium with angiogenic therapy, followed by cell transplant, would be more effective than the application of either strategy alone. Fischer 344 rats underwent left coronary artery ligation and injection of an adenovirus encoding VEGF 121, an empty expression cassette control vector, or saline solution. Capillary density in the infarcted region was determined in preliminary studies. Cardiomyocytes harvested from syngeneic Fischer rat fetuses were prelabeled and then injected directly into the infarct area 3 weeks after vector administration. Exercise treadmill testing was performed 2 weeks after cell transplantation, after which a cell viability index was calculated as the number of implanted (prelabeled) nuclei divided by the number of coadministered microspheres detected in sections of implanted myocardium. Capillary density in the area of infarction was significantly greater in adenovirus encoding VEGF 121 compared with rats injected with saline solution (P =.001). The cell survival index was also greater in adenovirus encoding VEGF 121 compared with animals injected with empty expression cassette control or saline solution (P =.0045). Exercise tolerance was nearly doubled in animals receiving adenovirus encoding VEGF 121 3 weeks prior to cell implantation compared with animals receiving adenovirus encoding VEGF 121 or cells alone or those receiving adenovirus encoding VEGF 121 at the time of cell implantation (P <.001). Pretreatment of an infarcted region of the heart with angiogenic mediators such as VEGF can enhance the efficacy of cellular cardiomyoplasty, presumably by creating a more favorable environment for the survival of transplanted cells.
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Ex vivo expanded endothelial progenitor cells (EPCs) represent a new potential approach for the revascularization of ischemic sites. However, local accumulation of infused EPCs in these sites is poor, and the mechanisms responsible for their homing are largely unknown. We observed the expression of L-selectin, an adhesion receptor that regulates lymphocyte homing and leukocyte rolling and migration, on ex vivo expanded blood-derived human EPCs. When EPCs were subcloned in SV40-T large Ag-transfected isolates, the copresence of L-selectin and endothelial lineage markers was confirmed. We therefore demonstrated that the expression of L-selectin by EPCs was functional because it mediates interaction with a murine endothelial cell line (H.end) expressing L-selectin ligands by way of transfection with alpha(1,3/4)-fucosyltransferase. Indeed, adhesion of EPCs after incubation at 4 degrees C on a rotating platform was enhanced on alpha(1,3/4)-fucosyltransferase-transfected H.end cells compared with control vector-transfected cells, and treatment with anti-L-selectin Abs prevented this event. We then studied the role of L-selectin in EPC homing in vivo. H.end cells were implanted s.c. in SCID mice to form endothelioma tumors, and EPCs were subsequently i.v. injected. L-selectin+ EPCs localized into alpha(1,3/4)-fucosyltransferase-transfected endothelial tumors to a greater extent than in control tumors, and they were able to directly contribute to tumor vascularization by forming L-selectin+ EPC-containing vessels. In conclusion, our results showed that a mechanism typical of leukocyte adhesion is involved in the vascular homing of EPCs within sites of selectin ligand expression. This observation may provide knowledge about the substrate to design strategies to improve EPC localization in damaged tissues.
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The mechanisms of homing of endothelial progenitor cells (EPCs) to sites of ischemia are unclear. Here, we demonstrate that ex vivo-expanded EPCs as well as murine hematopoietic Sca-1+/Lin- progenitor cells express beta2-integrins, which mediate the adhesion of EPCs to endothelial cell monolayers and their chemokine-induced transendothelial migration in vitro. In a murine model of hind limb ischemia, Sca-1+/Lin- hematopoietic progenitor cells from beta2-integrin-deficient mice are less capable of homing to sites of ischemia and of improving neovascularization. Preactivation of the beta2-integrins expressed on EPCs by activating antibodies augments the EPC-induced neovascularization in vivo. These results provide evidence for a novel function of beta2-integrins in postnatal vasculogenesis.
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Endothelial progenitor cells (EPCs) are bone marrow-derived cells that are augmented in response to ischemia and incorporated into neovascularization sites. We sought to determine whether circulating EPCs are related to collateral formation following non-ST segment elevation myocardial infarction (NSTEMI). Twenty patients who underwent percutaneous coronary intervention (PCI) within a week of NSTEMI were divided into two groups: patients without collaterals (coll-, n=10) and patients with Rentrop grade 3--4 collaterals (coll+, n=10). Blood samples were drawn before PCI and 24+/- 2 h after PCI. EPC colonies were grown from peripheral blood mononuclear cells, characterized, and counted. Using flow cytometry the percentage of cells co expressing vascular endothelial growth factor receptor-2 and CD 133 was determined. The coll+ group had higher degree of culprit vessel stenosis and lower initial thrombolysis in myocardial infarction flow grade. The relative number of EPCs before PCI was significantly higher in the coll+ group than in the coll- group (1.49 +/- 0.9% vs. 0.77+/- 0.4%, p= 0.045). There were no significant intergroup differences in the number of EPC colony-forming cells. The number of EPC colonies increased in the coll- group after PCI (9.5 +/- 4.8 to 14.0 +/- 5.9/10(6) cells, p=0.01). This study supports an association between circulating EPC levels and collateral formation in patients with an NSTEMI.
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The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitors, 'hemangiocytes,' constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4+ VEGFR1+ hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2-/- Csf3-/-), profound impairment in neovascularization was detected in sKitL-deficient Mmp9-/- as well as thrombocytopenic Thpo-/- and TPO receptor-deficient (Mpl-/-) mice. SDF-1-mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo-/-, Mpl-/- and Mmp9-/- mice. Transplantation of CXCR4+ VEGFR1+ hemangiocytes into Mmp9-/- mice restored revascularization, whereas inhibition of CXCR4 abrogated cytokine- and VEGF-A-mediated mobilization of CXCR4+ VEGFR1+ cells and suppressed angiogenesis. In conclusion, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+ VEGFR1+ hemangiocytes, whereas VEGFR1 is essential for their angiogenic competency for augmenting revascularization. Delivery of SDF-1 may be effective in restoring angiogenesis in individuals with vasculopathies.
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Clinical trials of bone marrow stem/progenitor cell therapy after myocardial infarction (MI) have shown promising results, but the mechanism of benefit is unclear. We examined the nature of endogenous myocardial repair that is dependent on the function of the c-kit receptor, which is expressed on bone marrow stem/progenitor cells and on recently identified cardiac stem cells. MI increased the number of c-kit+ cells in the heart. These cells were traced back to a bone marrow origin, using genetic tagging in bone marrow chimeric mice. The recruited c-kit+ cells established a proangiogenic milieu in the infarct border zone by increasing VEGF and by reversing the cardiac ratio of angiopoietin-1 to angiopoietin-2. These oscillations potentiated endothelial mitogenesis and were associated with the establishment of an extensive myofibroblast-rich repair tissue. Mutations in the c-kit receptor interfered with the mobilization of the cells to the heart, prevented angiogenesis, diminished myofibroblast-rich repair tissue formation, and led to precipitous cardiac failure and death. Replacement of the mutant bone marrow with wild-type cells rescued the cardiomyopathic phenotype. We conclude that, consistent with their documented role in tumorigenesis, bone marrow c-kit+ cells act as key regulators of the angiogenic switch in infarcted myocardium, thereby driving efficient cardiac repair.
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Umbilical cord blood (UCB) and bone marrow (BM)-derived stem and progenitor cells possess two characteristics required for successful tissue regeneration: extensive proliferative capacity and the ability to differentiate into multiple cell lineages. Within the normal BM and in pathological conditions, areas of hypoxia may have a role in maintaining stem cell fate or determining the fine equilibrium between their proliferation and differentiation. In this study, the transcriptional profiles and proliferation and differentiation potential of UCB CD133(+) cells and BM mesenchymal cells (BMMC) exposed to normoxia and hypoxia were analyzed and compared. Both progenitor cell populations responded to hypoxic stimuli by stabilizing the hypoxia inducible factor (HIF)-1alpha protein. Short exposures to hypoxia increased the clonogenic myeloid capacity of UCB CD133(+) cells and promoted a significant increase in BMMC number. The differentiation potential of UCB CD133(+) clonogenic myeloid cells was unaltered by short exposures to hypoxia. In contrast, the chondrogenic differentiation potential of BMMCs was enhanced by hypoxia, whereas adipogenesis and osteogenesis were unaltered. When their transcriptional profiles were compared, 183 genes in UCB CD133(+) cells and 45 genes in BMMC were differentially regulated by hypoxia. These genes included known hypoxia-responsive targets such as BNIP3, PGK1, ENO2, and VEGFA, and other genes not previously described to be regulated by hypoxia. Several of these genes, namely CDTSPL, CCL20, LSP1, NEDD9, TMEM45A, EDG-1, and EPHA3 were confirmed to be regulated by hypoxia using quantitative reverse transcriptase polymerase chain reaction. These results, therefore, provide a global view of the signaling and regulatory network that controls oxygen sensing in human adult stem/progenitor cells derived from hematopoietic tissues.
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Neovascularization plays a critical role in the growth and metastatic spread of tumors and involves recruitment of circulating endothelial progenitor cells (EPC) from bone marrow as well as sprouting of preexisting endothelial cells. In this study, we examined if EPCs could promote tumor angiogenesis and would be an effective cellular target for an angiogenesis inhibitor, the recombinant kringle domain of tissue-type plasminogen activator (TK1-2). When TK1-2 was applied in the ex vivo culture of EPCs isolated from human cord blood, TK1-2 inhibited adhesive differentiation of mononuclear EPCs into endothelial-like cells. In addition, it inhibited the migration of ex vivo cultivated EPCs and also inhibited their adhesion to fibronectin matrix or endothelial cell monolayer. When A549 cancer cells were coimplanted along with ex vivo cultivated EPCs s.c. in nude mice, the tumor growth was increased. However, the tumor growth and the vascular density of tumor tissues increased by coimplanted EPCs were decreased upon TK1-2 treatment. Accordingly, TK1-2 treatment reduced the remaining number of EPCs in tumor tissues and their incorporation into the host vascular channels. In addition, overall expression levels of vascular endothelial growth factor (VEGF) and von Willebrand factor in tumor tissues were decreased upon TK1-2 treatment. Interestingly, strong VEGF expression by implanted EPCs was decreased by TK1-2. Finally, we confirmed in vitro that TK1-2 inhibited VEGF secretion of EPCs. TK1-2 also inhibited endothelial cell proliferation and migration induced by the conditioned medium of EPCs. Therefore, we concluded that EPCs, as well as mature endothelial cells, could be an important target of TK1-2.
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The results from small clinical studies suggest that therapy with adult bone marrow (BM)-derived cells (BMCs) reduces infarct size and improves left ventricular function and perfusion. However, the effects of BMC transplantation in patients with ischemic heart disease remains unclear. We searched MEDLINE, EMBASE, Science Citation Index, CINAHL (Cumulative Index to Nursing and Allied Health), and the Cochrane Central Register of Controlled Trials (CENTRAL) (through July 2006) for randomized controlled trials and cohort studies of BMC transplantation to treat ischemic heart disease. We conducted a random-effects meta-analysis across eligible studies measuring the same outcomes. Eighteen studies (N = 999 patients) were eligible. The adult BMCs included BM mononuclear cells, BM mesenchymal stem cells, and BM-derived circulating progenitor cells. Compared with controls, BMC transplantation improved left ventricular ejection fraction (pooled difference, 3.66%; 95% confidence interval [CI], 1.93% to 5.40%; P<.001); reduced infarct scar size (-5.49%; 95% CI, -9.10% to -1.88%; P = .003); and reduced left ventricular end-systolic volume (-4.80 mL; 95% CI, -8.20 to -1.41 mL; P = .006). The available evidence suggests that BMC transplantation is associated with modest improvements in physiologic and anatomic parameters in patients with both acute myocardial infarction and chronic ischemic heart disease, above and beyond conventional therapy. Therapy with BMCs seems safe. These results support conducting large randomized trials to evaluate the impact of BMC therapy vs the standard of care on patient-important outcomes.
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Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.
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Noncellular differentiation effects have emerged as important mechanisms mediating therapeutic effects of stem or progenitor cell transplantation. Here, we investigated the expression patterns and sources of humoral factors and their regional and systemic biological effects after bone marrow (BM)-derived endothelial progenitor cell (EPC) transplantation into ischemic myocardium. Although most of the transplanted EPCs disappeared within a week, up-regulation of multiple humoral factors was sustained for longer than two weeks, which correlated well with the recovery of cardiac function. To determine the source of the humoral factors, we injected human EPCs into immunodeficient mice. Whereas the expression of human EPC (donor)-derived cytokines rapidly decreased to a nondetectable level within a week, up-regulation of mouse (recipient)-derived cytokines, including factors that could mobilize BM cells, was sustained. Histologically, we observed higher capillary density, a higher proliferation of myocardial cells, a lower cardiomyocyte apoptosis, and reduced infarct size. Furthermore, after EPC transplantation, BM-derived stem or progenitor cells were increased in the peripheral circulation and incorporated into the site of neovascularization and myocardial repair. These data indicate that myocardial EPC transplantation induces humoral effects, which are sustained by host tissues and play a crucial role in repairing myocardial injury.
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Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-kit-positive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1α, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate de novo coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium. • coronary blood flow • infarct size • myocardial regeneration • stem cells • vasculogenesis
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This study investigated the factors responsible for migration and homing of magnetically labeled AC133(+) cells at the sites of active angiogenesis in tumor. AC133(+) cells labeled with ferumoxide-protamine sulfate were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. An MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB), platelet-derived growth factor (PDGF), hypoxia-inducible factor-1alpha (HIF-1alpha), stromal cell derived factor-1 (SDF-1), matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and endothelial markers. Fresh snap-frozen strips from the central and peripheral parts of the tumor were collected for Western blotting. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB(+)/AC133(+) cells were positive for endothelial cells markers. At the sites of PB(+)/AC133(+) cells, both HIF-1alpha and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB(+)/AC133(+) cell localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1, MMP-2, and PDGF at the peripheral parts of the tumors. HIF-1alpha was expressed at both the periphery and central parts of the tumor. This work demonstrates that magnetically labeled cells can be used as probes for MRI and histological identification of administered cells.
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Stem cells are the building blocks through which tissues are developed and maintained. We and many other groups predict that stem cells will prove tremendously useful in clinical medicine. Possible uses include systems for high- throughput drug screens, as in vitro models of disease and, eventually, in treating diseases associated with cell defi- ciency. As one of the least regenerative organs in the body, the heart stands to benefit greatly from addition of new parenchymal cells. Cardiovascular researchers have risen to this challenge and, as a result, cardiac repair is arguably the most advanced program in the emerging field of regenera- tive medicine. Progress in this field has been rapid, from humble beginnings with committed skeletal (1-3) or cardiac muscle cells (4-6), moving to multipotent adult stem cells (7-9) and, most recently, to embryonic stem cells (10-13). In this brief commentary, we review some important recent developments in stem cell-based tissue repair. (Read- ers wishing additional basic and clinical information are directed to several recent in-depth reviews (14-16) on the field.) Like other areas involving stem cell-based regeneration, the field of cardiac repair has its share of controversies. These will also be touched upon, with an aim of separating experi- mental observation, on which there is much agreement, from interpretation, which varies widely at the moment.
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Neovascularization by endothelial progenitor cells (EPC) for the treatment of ischaemic diseases has been a topic of intense research. The CD34+ cell is often designated as EPC, because it contributes to repair of ischaemic injuries through neovascularization. However, incorporation of CD34+ cells into the neovasculature is limited, suggesting another role which could be paracrine. CD14+ cells can also differentiate into endothelial cells and contribute to neovascularization. However, the low proliferative capacity of CD14+ cell-derived endothelial cells hampers their use as therapeutic cells. We made the assumption that an interaction between CD34+ and CD14+ cells augments endothelial differentiation of the CD14+ cells. In vitro, the influence of CD34+ cells on the endothelial differentiation capacity of CD14+ cells was investigated. Endothelial differentiation was analysed by expression of endothelial cell markers CD31, CD144, von Willebrand Factor and endothelial Nitric Oxide Synthase. Furthermore, we assessed proliferative capacity and endothelial cell function of the cells in culture. In monocultures, 63% of the CD14+-derived cells adopted an endothelial cell phenotype, whereas in CD34+/CD14+ co-cultures 95% of the cells showed endothelial cell differentiation. Proliferation increased up to 12% in the CD34+/CD14+ co-cultures compared to both monocultures. CD34-conditioned medium also increased endothelial differentiation of CD14+ cells. This effect was abrogated by hepatocyte growth factor neutralizing antibodies, but not by interleukin-8 and monocyte chemoattractant protein-1 neutralizing antibodies. We show that co-culturing of CD34+ and CD14+ cells results in a proliferating population of functional endothelial cells, which may be suitable for treatment of ischaemic diseases such as myocardial infarction.
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Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. Specifically, three carbohydrate binding proteins termed selectins (E-, P-, and L-selectin) have been shown to be essential for leukocyte rolling along the vascular endothelium, the first step in the recruitment of leukocytes from the blood into inflammatory sites or into secondary lymphoid organs. Although this phenomenon is well-established, little is known about the molecular-level interactions on which it depends. All three selectins recognize sulfated and sialylated derivatives of the Lewis x [Le(x):Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and Lewis a [Le(a): Gal beta 1-->3(Fuc alpha 1-->4)GlcNAc] trisaccharide cores with affinities in the millimolar range, and it is believed that variants of these structures are the carbohydrate determinants of selectin recognition. Recently it was shown that the mucin GlyCAM-1, a secreted physiological ligand for L-selectin, is capped with sulfated derivatives of sialyl Lewis x [sLe(x): Sia alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and that sulfation is required for the high-affinity interaction between GlyCAM-1 and L-selectin. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Our results suggest that 6-sulfo sLe(x) binds to L-selectin with higher affinity than does sLe(x) or 6'-sulfo sLe(x) and that sulfation of sLe(x) capping groups on GlyCAM-1 at the 6-position is important for L-selectin recognition.
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A device based on the cone-and-plate flow geometry commonly employed for viscometry was developed for the investigation of cell-surface interactions. The cone-and-plate geometry is capable of generating uniform, constant shear-rate flow fields, and control of cone rotational speed allows for easy variation of fluid shear rate. The current design is adapted for use with any material that is available in the form of a flat plate (film or coating). It also allows for replicate samples (the same or different surfaces) to be evaluated simultaneously. The device was tested under varying flow conditions for its ability to measure platelet adhesion from suspensions of washed platelets containing red cells. Collagen- and albumin-coated polymer materials were used as "standard" surfaces of known platelet reactivity (high and low, respectively). Adhesion to the collagen-coated surface was measured over a range of shear rate from 0 to 300 s(-1) and times up to 15 min. Platelet adhesion was observed to increase with increasing shear rate and time. Adhesion was significantly higher in the presence of red cells as has been observed by others. Effective platelet diffusion coefficients, calculated from the data on adhesion to the collagen surface, increased with increasing shear rate. Very little platelet adhesion to the albumin-coated surface, known to be unreactive to platelets, was observed when measured over a 15 min time period at 300 s(-1) shear rate, indicating that the device itself does not stimulate the platelets in the flow field. The data generated provide validation for this device as a simple means of measuring cell adhesion under controlled flow conditions to any smooth surface available in flat plate form.
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L-selectin is a cell surface glycoprotein expressed on most leukocyte subsets that mediates leukocyte interaction with ligands on lymphoid tissue high endothelial venule cells as well as with ligands on activated endothelium at sites of inflammation in nonlymphoid organs. Similar to two other members of the selectin family, L-selectin behaves as a lectin, recognizing carbohydrate ligands in a calcium-dependent fashion. Recent in vivo studies reveal the importance of L-selectin expression in both normal and pathologic conditions.
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L-selectin has been shown to be important in mediating leukocyte recruitment during inflammatory responses. Although there are numerous in vitro studies demonstrating that engagement of L-selectin leads to the activation of several signaling pathways potentially contributing to subsequent adhesion, emigration, or even migration through the interstitium, whether this actually induces cellular events in vivo is completely unknown. Therefore, we used intravital microscopy to visualize the role of L-selectin in downstream leukocyte adhesion, emigration, and interstitial migration events in wild-type and L-selectin-deficient (L-selectin(-/-)) mice. The cremaster muscle was superfused with the chemotactic inflammatory mediators platelet-activating factor or KC. Leukocyte rolling, adhesion, and emigration in postcapillary venules were examined, and the migration of emigrated leukocytes was recorded continuously using time-lapse videomicroscopy. Platelet-activating factor increased leukocyte adhesion to a similar level in both wild-type and L-selectin(-/-) mice. In contrast, both the number of emigrated leukocytes and the distance of extravascular migration were significantly reduced in L-selectin(-/-) mice. A similar pattern was observed in response to the superfusion of KC. Because superfusion of these mediators induced chemokinesis, we developed a new in vivo chemotaxis assay using slow release of KC from an agarose gel positioned 350 microm from a postcapillary venule. These experiments showed that L-selectin(-/-) leukocytes were also severely impaired in their ability to respond to a directional cue. These findings indicate that L-selectin is important in enabling leukocytes to respond effectively to chemotactic stimuli in inflamed tissues.
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Infusion of different hematopoietic stem cell populations and ex vivo expanded endothelial progenitor cells augments neovascularization of tissue after ischemia and contributes to reendothelialization after endothelial injury, thereby, providing a novel therapeutic option. However, controversy exists with respect to the identification and the origin of endothelial progenitor cells. Overall, there is consensus that endothelial progenitor cells can derive from the bone marrow and that CD133/VEGFR2 cells represent a population with endothelial progenitor capacity. However, increasing evidence suggests that there are additional bone marrow-derived cell populations (eg, myeloid cells, "side population" cells, and mesenchymal cells) and non-bone marrow-derived cells, which also can give rise to endothelial cells. The characterization of the different progenitor cell populations and their functional properties are discussed. Mobilization and endothelial progenitor cell-mediated neovascularization is critically regulated. Stimulatory (eg, statins and exercise) or inhibitory factors (risk factors for coronary artery disease) modulate progenitor cell levels and, thereby, affect the vascular repair capacity. Moreover, recruitment and incorporation of endothelial progenitor cells requires a coordinated sequence of multistep adhesive and signaling events including adhesion and migration (eg, by integrins), chemoattraction (eg, by SDF-1/CXCR4), and finally the differentiation to endothelial cells. This review summarizes the mechanisms regulating endothelial progenitor cell-mediated neovascularization and reendothelialization.
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Adult stem cells can contribute to myocardial regeneration after ischemic injury. Bone marrow and skeletal muscles contain a population of CXCR4+ cells expressing genes specific for muscle progenitor cells that can be mobilized into the peripheral blood. The aims of the study were (1) to confirm the presence of early tissue-committed cells expressing cardiac, muscle, and endothelial markers in populations of mononuclear cells in peripheral blood and (2) to assess the dynamics and magnitude of the mobilization of CD34+, CD117+, CXCR4+, c-met+, CD34/CD117+, and CD34/CXCR4+ stem cells into peripheral blood in relation to inflammatory and hematopoietic cytokines in patients with ST-segment-elevation acute myocardial infarction (STEMI). Fifty-six patients with STEMI (<12 hours), 39 with stable angina, and 20 healthy control subjects were enrolled. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for detection of tissue-specific markers. The number of the cells was assessed by use of a flow cytometer on admission, after 24 hours, and after 7 days. RT-PCR revealed increased expression of mRNA (up to 3.5-fold increase) for specific cardiac (GATA4, MEF2C, Nkx2.5/Csx), muscle (Myf5, Myogenin, MyoD), and endothelial (VE-cadherin, von Willebrand factor) markers in peripheral blood mononuclear cells. The number of CD34/CXCR4+ and CD34/CD117+ and c-met+ stem cells in peripheral blood was significantly higher in STEMI patients than in stable angina and healthy subjects, peaking on admission, without further significant increase after 24 hours and 7 days. The study demonstrates in the setting of STEMI a marked mobilization of mononuclear cells expressing specific cardiac, muscle, and endothelial markers as well as CD34/CXCR4+ and CD34/CD117+ and c-met+ stem cells and shows that stromal cell-derived factor-1 is an important factor influencing the mobilization.
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Therapeutic angiogenesis aims at restoring perfusion to chronically ischemic myocardial territories by using growth factors or cells, without intervening on the epicardial coronary arteries. Despite angiogenesis having received considerable scientific attention over the last decade, it has not yet been shown to provide clinical benefit and is still reserved for patients who have failed conventional therapies. Nevertheless, angiogenesis is a very potent physiologic process involved in the growth and development of every animal and human, and it is likely that its use for therapeutic purposes, once its underlying mechanistic basis is better understood, will one day become an important modality for patients with CAD and other types of organ ischemia. This review summarizes current knowledge in therapeutic angiogenesis research.
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Mobilization of endogenous endothelial progenitor cells (EPCs) from the bone marrow may be an alternative way to increase neovascularization and may be used as therapeutic option for the treatment of ischemic cardiovascular diseases. In this review, we discuss the EPC mobilizing effects of pro-inflammatory cytokines such as granolocyte monocyte colony-stimulating factor and granulocyte colony-stimulating factor, growth factors such as vascular endothelial growth factor, placental growth factor, erythropoietin, and angiopoietin-1, chemokines such as stromal cell-derived factor-1, hormones such as estrogens and lipid-lowering and anti-diabetic drugs, as well as physical activity.
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Therapeutic angiogenesis has yielded promising results in animal models, including the demonstration of newly created blood vessels, increased perfusion and functional benefits. On the other hand, clinical studies using similar methods of angiogenesis have so far been disappointing. The possibility that endothelial dysfunction may play a role in this bench-to-bedside discrepancy has led to further research on the role of endothelial-derived mediators in the angiogenic cascade. One of these mediators is nitric oxide (NO), which plays an integral role in the development and maintenance of a microvascular network and whose local availability is altered in endothelial dysfunction. This article outlines the role of NO in the angiogenic response and discusses possible therapeutic options to optimise endothelial dysfunction and NO availability in patients undergoing angiogenic therapy.
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Intracoronary transfer of autologous bone marrow cells (BMCs) promotes recovery of left ventricular systolic function in patients with acute myocardial infarction. Although the mechanisms of this effect remain to be established, homing of BMCs into the infarcted myocardium is probably a critical early event. We determined BMC biodistribution after therapeutic application in patients with a first ST-segment-elevation myocardial infarction who had undergone stenting of the infarct-related artery. Unselected BMCs were radiolabeled with 100 MBq 2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) and infused into the infarct-related coronary artery (intracoronary; n=3 patients) or injected via an antecubital vein (intravenous; n=3 patients). In 3 additional patients, CD34-positive (CD34+) cells were immunomagnetically enriched from unselected BMCs, labeled with 18F-FDG, and infused intracoronarily. Cell transfer was performed 5 to 10 days after stenting. More than 99% of the infused total radioactivity was cell bound. Nucleated cell viability, comparable in all preparations, ranged from 92% to 96%. Fifty to 75 minutes after cell transfer, all patients underwent 3D PET imaging. After intracoronary transfer, 1.3% to 2.6% of 18F-FDG-labeled unselected BMCs were detected in the infarcted myocardium; the remaining activity was found primarily in liver and spleen. After intravenous transfer, only background activity was detected in the infarcted myocardium. After intracoronary transfer of 18F-FDG-labeled CD34-enriched cells, 14% to 39% of the total activity was detected in the infarcted myocardium. Unselected BMCs engrafted in the infarct center and border zone; homing of CD34-enriched cells was more pronounced in the border zone. 18F-FDG labeling and 3D PET imaging can be used to monitor myocardial homing and biodistribution of BMCs after therapeutic application in patients.
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The maintenance of endothelial integrity plays a critical role in preventing atherosclerotic disease progression. Endothelial progenitor cells (EPCs) were experimentally shown to incorporate into sites of neovascularization and home to sites of endothelial denudation. Circulating EPCs may thus provide an endogenous repair mechanism to counteract ongoing risk factor-induced end