Combinatorial approach for improving the outcome of angiogenic therapy in ischemic tissues

Two major populations of endothelial progenitor cells (EPC), namely endothelial colony forming cells (ECFC, or late outgrowth EPC) and circulating angiogenic cells (CAC, or early outgrowth EPC) have been reported to play important roles in vasculogenesis in numerous pathological conditions. However, the poor retention of cells into the ischemic tissue and neovessel fragility are two major flaws that need to be overcome for successful angiogenic therapy. The objective of this study was to explore and exploit the functional properties of EPC populations in order to increase the effectiveness of post-ischemic cell therapy. The results indicate different, still complementary, effects of the two EPC populations on adherence and proliferation of vascular endothelial cells. Matrigel plug assay and mouse hind limb ischemia model showed that concomitant administration of CAC-secreted factors and ECFC resulted in three-fold increase in local cell retention and improved muscle perfusion, vessel maturation and hind limb regeneration, in comparison to either treatment alone. By concluding, factors secreted by CAC co-administered at the time of ECFC transplantation improve tissue regeneration and vascular repair through stabilization of newly-derived blood vessels.     

Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis.

The formation of new blood vessels proceeds by both vasculogenesis and angiogenesis. The development of models, which fully recapitulate spatio-temporal events involved during these processes, are crucial to fully understand their mechanisms of regulation. In vitro differentiation of murine embryonic stem (ES) cells has been shown to be a useful tool to investigate factors and genes potentially involved in vasculogenesis (Hirashima et al, 1999; Risau et al, 1988; Vittet et al, 1996; Wang et al, 1992; Wartenberg et al, 1998). We asked here whether this model system can also recapitulate angiogenesis, which may offer new means to study mechanisms involved in this process. ES-derived embryoid bodies (EBs) obtained after 11 days of differentiation, in which a primitive vascular network had formed, were then subcultured into a type I collagen matrix. In the presence of angiogenic growth factors, EBs rapidly developed branching pseudopods. Whole mount immunostainings with a PECAM antibody revealed that more than 75% EBs displayed, within a few days, a large number of endothelial outgrowths that can give tube-like structures with concomitant differentiation of alpha-smooth muscle actin positive cells, thus evoking sprouting angiogenesis. High expression levels of flk1 (VEGFR2), flt1 (VEGFR1), tie-1, and tie-2 are also found, indicating that budding endothelial cells displayed an angiogenic phenotype. The endothelial sprouting response was specifically induced by angiogenic factors with a major contribution of vascular endothelial growth factor (VEGF). Known angiostatic agents, such as platelet factor 4 (PF4), angiostatin, and endostatin inhibited the formation of endothelial sprouts induced by angiogenic factors. Moreover, consistent with the in vivo phenotype, VE-cadherin deficient EBs failed to develop angiogenesis in this model. ES cell differentiation can then recapitulate, in addition to vasculogenesis, the early stages of sprouting angiogenesis. This model system, in which genetic modifications can be easily introduced, may be of particular interest to investigate unsolved questions and molecular mechanisms involved in blood vessel formation.     

EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury

The endothelium of the adult vasculature is normally quiescent, with the exception of the vasculature of the female reproductive system. However, in response to appropriate stimuli (ie, wound healing, atherosclerosis, tumor growth and metastasis, arthritis) the vasculature becomes activated and grows new capillaries through angiogenesis. We have recently identified a novel endothelial-restricted gene, Egfl7, that encodes a 41-kd secreted protein (Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H: Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn 2004, 230:316-324). Egfl7 is expressed at high levels early during mouse embryonic development and is strictly associated with the vascular bed. In this study, we investigated Egfl7 expression in the quiescent adult vasculature, in the pregnant uterus, and in two different models of arterial injury, namely ballooning and ferric chloride injury. By RNA in situ hybridization, Egfl7 expression in the vasculature was found to be restricted to the endothelium of the capillaries and mature vessels. In the pregnant uterus, increased vascularization was accompanied by up-regulation of Egfl7. On arterial injury, Egfl7 expression was up-regulated in the regenerating endothelium, but not in the neointima. Importantly, the EGFL7 protein acted as a chemoattractant for embryonic endothelial cells and fibroblasts in a cell migration assay. Together, these results suggest that Egfl7 functions in the formation and maintenance of endothelial integrity and that its up-regulation may be a critical component in the reorganization of the vascular bed in response to angiogenic stimuli.     

Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization

A subset of human peripheral blood mononuclear cells (PB-MNCs) differentiate into endothelial progenitor cells (EPCs) that participate in postnatal neovascularization. Although tissue ischemia can mobilize EPCs from bone marrow, the effects of hypoxia on differentiation and angiogenic function of EPCs are little known. We examined whether hypoxic conditioning would modulate differentiation and function of human PB-MNC-derived EPCs. A subset of PB-MNCs gave rise to EPC-like attaching (AT) cells under either normoxic or hypoxic conditions. However, hypoxia much enhanced the differentiation of AT cells from PB-MNCs compared with normoxia. AT cells released vascular endothelial growth factor (VEGF) protein and expressed CD31 and kinase insert domain receptor/VEGFR-2, endothelial lineage markers, on their surface, which were also enhanced by hypoxia. Both a neutralizing anti-VEGF mAb and a KDR-specific receptor tyrosine kinase inhibitor, SU1498, suppressed PB-MNC differentiation into EPC-like AT cells in a dose-dependent manner. Migration of AT cells in response to VEGF as examined by a modified Boyden chamber apparatus was also enhanced by hypoxia. Finally, in vivo neovascularization efficacy was significantly enhanced by in vitro hypoxic conditioning of AT cells when cells were transplanted into the ischemic hindlimb of immunodeficient nude rats. In conclusion, hypoxia directly stimulated differentiation of EPC-like AT cells from human PB-MNC culture. Moreover, hypoxic preconditioning of AT cells before in vivo transplantation is a useful means to enhance therapeutic vasculogenesis.     

VE-statin/Egfl7 siRNA inhibits angiogenesis in malignant glioma in vitro

This study investigated the role of VE-statin/Egfl7 and its mechanism in angiogenesis in malignant glioma. Transwell culture plates were used to establish an U251-HUVEC co-culture system, which was used to mimic the interaction between malignant glioma and endothelial cells. Lentiviral vectors expressing VE-statin/Egfl7 siRNA were constructed, and U251 cells and HUVECs were transfected to inhibit VE-statin/Egfl7 expression. The proliferation, adherence, migration, and lumen formation of endothelial cells were assayed to investigate the influence of VE-statin/Egfl7 on angiogenesis in malignant glioma in vitro. Data showed that HUVEC growth was temporarily slowed after silencing the VE-statin/Egfl7 gene but rapidly returned to normal. Although endothelial cell migration was not influenced, cell adherence was markedly inhibited. Furthermore, the endothelial cells failed to generate a capillary-like lumen after VE-statin/Egfl7 gene silencing. Therefore, it can be concluded that VE-statin/Egfl7 may regulate the adherence of endothelial cells, thus playing an important role in endothelium-induced lumen formation during angiogenesis in malignant glioma.     

VE-statin/Egfl7 expression in malignant glioma and its relevant molecular network

This study investigated VE-statin/Egfl7 expression and its role and regulatory mechanism in malignant glioma progression. Forty-five paraffin-embedded glioma (grade I-II: n=24; grade III-IV: n=21) were examined. VE-statin/Egfl7 protein expression was detected via immunohistochemistry, and its correlation with pathological grade was evaluated. Three-dimensional cell culture was then performed to investigate the influence of VE-statin/Egfl7 on the angiogenesis of umbilical vein endothelial cells. Microarray detection was used to molecularly profile VE-statin/Egfl7 and relevant signaling pathways in malignant glioma (U251 cells). Data showed that VE-statin/Egfl7 protein was mainly expressed in the cytoplasm of cancer and vascular endothelial cells and was significantly related to the degree of malignancy (t=4.399, P<0.01). Additionally, VE-statin/Egfl7 expression was low in certain gray-matter neurons but undetectable in glial cells. VE-statin/Egfl7 gene silencing significantly inhibited angiogenesis in umbilical vein endothelial cells. The following microarray results were observed in VE-statin/Egfl7-silenced U251 cells: 1) EGFR family members showed the highest differential expression, accounting for 5.54% of differentially expressed genes; 2) cell survival-related signaling pathways changed significantly; and 3) the integrin ανβ3 signaling pathway was markedly altered. Thus, malignant glioma cells and glioma vascular endothelial cells highly express VE-statin/Egfl7, which is significantly correlated with the degree of malignancy. Moreover, VE-statin/Egfl7 plays an important role in glioma angiogenesis. Microarray results indicate that VE-statin/Egfl7 may regulate EGFR and integrins to influence the FAK activity of downstream factors, triggering the PI3K/Akt and Ras/MAPK cascades and subsequent malignant glioma development.     

Angiogenesis: basic and clinical aspects

The cardiovascular system is the first functional organ system to develop in the vertebrate embryo. A widely accepted view is that blood vessels arise through two mechanisms during development, vasculogenesis and angiogenesis. New vessels in the adult arise mainly through angiogenesis, although vasculogenesis also may occur. The existence of a postnatal vasculogenesis is also supported by the evidence that both endothelial cells and endothelial precursor cells co-exist in the circulation. Angiogenesis is a biological process by which new capillaries are formed and it occurs in many physiological and pathological conditions. It is controlled by the net balance between molecules that have positive and negative regulatory activity and this concept had led to the notion of the "angiogenic switch", depending on an increased production of one or more of the positive regulators of angiogenesis. Considerable benefit can be derived in the clinical setting from manipulating angiogenesis, either positively or negatively. There is a variety of important clinical situations in which it would be desiderable to promote angiogenic processes, such as situations in which it would be desiderable to promote angiogenic processes, such as for the induction of collateral vascularization in an ischemic heart or limb. Conversely, there are pathologic conditions in which preventing angiogenic processes could be useful in the treatment of a growing tumor or a chronic inflammatory process.     

Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior

One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.     

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.     

Bioceramic-mediated trophic factor secretion by mesenchymal stem cells enhances in vitro endothelial cell persistence and in vivo angiogenesis

Mesenchymal stem cells (MSCs) seeded in composite implants formed of hydroxyapatite (HA) and poly (lactide-co-glycolide) (PLG) exhibit increased osteogenesis and enhanced angiogenic potential. Endothelial colony-forming cells (ECFCs) can participate in de novo vessel formation when implanted in vivo. The aim of this study was to determine the capacity of HA-PLG composites to cotransplant MSCs and ECFCs, with the goal of accelerating vascularization and resultant bone formation. The incorporation of HA into PLG scaffolds improved the efficiency of cell seeding and ECFC survival in vitro. We observed increases in mRNA expression and secretion of potent angiogenic factors by MSCs when cultured on HA-PLG scaffolds compared to PLG controls. Upon implantation into an orthotopic calvarial defect, ECFC survival on composite scaffolds was not increased in the presence of MSCs, nor did the addition of ECFCs enhance vascularization beyond increases observed with MSCs alone. Microcomputed tomography (micro-CT) performed on explanted calvarial tissues after 12 weeks revealed no significant differences between treatment groups for bone volume fraction (BVF) or bone mineral density (BMD). Taken together, these results provide evidence that HA-containing composite scaffolds seeded with MSCs can enhance neovascularization, yet MSC-secreted trophic factors do not consistently increase the persistence of co-transplanted ECFCs.     

Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells.

We report the cloning and characterization of a novel epidermal growth factor (EGF) domain gene that was identified in a retroviral gene entrapment screen and is expressed in endothelial cells. This gene encodes a protein of 278 amino acids with an amino-terminal signal peptide and two centrally located EGF-like domains. We have named this novel gene in accordance with the guidelines of the Mouse Genome Informatics group Egfl7, for EGF-like domain 7. Egfl7 mRNA is expressed in highly vascularized adult tissues such as the lung, heart, uterus, and ovary. In addition, Egfl7 is expressed early during mouse embryogenesis and in undifferentiated murine embryonic stem cells. The analysis of Egfl7 expression in embryonic day 9.5 embryos by in situ hybridization indicates that Egfl7 is expressed in vascular structures in both the embryo proper and the yolk sac and at sites of mesodermal precursors of angioblasts. Within the cell, EGFL7 protein is localized to the endoplasmic reticulum and Golgi apparatus, suggesting that the protein is targeted for secretion. Indeed, recombinant EGFL7 is readily detectable in the supernatant media of transiently transfected HEK293 cells. We also report the identification of an Egfl7 paralog, Egfl8, and show that EGFL8 protein shares similar domains and molecular weight with EGFL7.     

Nicotine enlivenment of blood flow recovery following endothelial progenitor cell transplantation into ischemic hindlimb

Nicotine has been demonstrated to stimulate postnatal angiogenesis, having an antiapoptotic effect on endothelial cells. Given the extent of this angiogenesis-promoting effect, we hypothesized that nicotine may also stimulate postnatal vasculogenesis on endothelial progenitor cells (EPCs). Our analyses reveal some intriguing results using an in vitro assay with 2 x 10(-6) M of nicotine (smoker's average nicotine concentration and the dose of nicotine replacement therapy). The proliferation and migration activities of human EPCs cultured from peripheral blood mononuclear cells of non-smoking healthy volunteers were not affected by nicotine. The effect of nicotine on EPC survival was significantly enhanced under serum starvation on the ratio of Hoechest 33342-stained pyknotic nuclear cells as well as Annexin-V-stained cells to total cells. Furthermore, the antiapoptotic effect of nicotine was blocked completely by nicotinic acetylcholine receptor (nAChR) antagonist hexamethonium. Next, we verified how nicotine acts in vivo. Nicotine (100 ng/ml) was administered orally for 7 days before and 4 weeks after injection of cultured EPCs (1 x 10(5) /mouse) into the tail veins of 8-week-old athymic nude mice with ischemic hindlimbs. Laser doppler imaging analysis indicated that blood perfusion in the ischemic hindlimb was significantly enhanced in EPCs plus nicotine, as compared with EPCs alone. These findings suggest nicotine improves blood flow following EPC transplantation in patients with ischemic diseases.     

Modulation of angiogenic functions in human macrophages by biomaterials

We examined the ability of polyvinylchloride (PVC), polytetrafluorethylene (PTFE) and tissue culture polystyrene (TCPS) to affect angiogenic functions in human monocyte-derived macrophages by measuring the mRNA expression of genes encoding angiogenic and anti-angiogenic molecules including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and thrombospondin-1 (Tsp-1). The angiogenic activity of the corresponding macrophage conditioned media (CM) was measured by the proliferation of endothelial cells and the sprouting of new capillaries from fragments of human placental blood vessels. We determined that bFGF was not expressed in macrophages while VEGF and Tsp-1 mRNAs were expressed constitutively. Ang-1 was expressed in macrophages cultured up to 7 days on PTFE and TCPS independent of the culture stage. In contrast, macrophages cultured on PVC did not produce detectable amounts of Ang-1 mRNA after 7 days. CM from macrophages cultured either on PTFE or TCPS stimulated angiogenesis whereas CM from macrophages cultured on PVC inhibited it. The results demonstrate that polymers can cause differential expression of the angiogenic molecule Ang-1 in macrophages. They also induce different phenotypes of macrophages, which can either stimulate or inhibit angiogenesis suggesting a material-dependent influence on neovascularization.     

Human embryonic stem cells as an in vitro model for human vascular development and the induction of vascular differentiation

Early embryonic blood vessels are typically composed of fragile tubes of endothelial cells encircled by vascular smooth muscle cells. Early human vasculogenesis was explored in spontaneous and directed differentiation models derived from human embryonic stem (HES) cells. In a 3-dimensional (3D) model, HES cells were studied for their potential for vascular differentiation during the spontaneous formation of embryoid bodies. Directed differentiation was investigated by means of a 2-dimensional (2D) differentiation method to promote vascular differentiation from HES cells (without the formation of embryoid bodies). Using this latter approach, up-regulation of early lineage markers of endothelial progenitors were induced. Additional culture under strict conditions and exposure to angiogenic growth factors resulted in a prolonged differentiation pathway into mature endothelial cells and up-regulation of vascular smooth muscle cell markers. The use of 3D collagen gels and Matrigel assays for the induction and inhibition of human vascular sprouting in vitro further established the vascular potential of the cells generated by the 2D differentiation system. Our study shows that HES cells can provide useful models to study early differentiation and development of blood vessels. Moreover, the 2D differentiation model facilitates both the production of vascular lineage cells from HES cells for various potential therapeutic applications and also provides a model for studying the mechanisms involved in early human embryonic blood vessel development.     

Cellular players in angiogenesis during the course of systemic sclerosis

Vascular endothelial injury in Systemic Sclerosis (SSc) leads to pathological changes in the blood vessels that adversely impact the physiology of many organs, resulting in chronic tissue ischemia. The response to hypoxia induces complex cellular and molecular mechanisms in the attempt to recover endothelial cell function and tissue perfusion. The progressive losses of capillaries on one hand, and the vascular remodeling of arteriolar vessels on the other, result in insufficient blood flow, causing severe and chronic hypoxia. Hypoxia is a major stimulus of angiogenesis, leading to the expression of pro-angiogenic molecules, mainly of Vascular Endothelial Growth Factor (VEGF), which triggers the angiogenic process. Nevertheless, in SSc patients there is no evidence of adaptive angiogenesis. Failure of the angiogenic process in SSc largely depends on alteration in the balance between pro- and anti-angiogenic factors, as well as on functional alterations of the cellular players involved in the angiogenic and vasculogenic program. A decreased urokinase plasminogen activator (uPA) dependent invasion, proliferation, and capillary morphogenesis, was showed in SSc endothelial cells (EC). Although hematopoietic endothelial progenitor cells (EPC) count in the peripheral blood of SSc patients is still a matter of controversy, alterations in mobilization process, an excessive immune-mediated EPC destruction in the peripheral circulation or in the bone marrow, a progressive depletion of EPCs following homing to ischemic tissues under persistent peripheral vascular injury, an intrinsic functional impairment could lead to poor vasculogenesis. Human mesenchymal stem cells represent an alternative source of endothelial progenitor cells and it has been observed that their angiogenic potential is reduced in SSc. Targeting autologous stem and progenitor cells could be an ideal tool to counteract and repair dysfunctional angiogenesis.     

Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

To elucidate the role of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, in tumour angiogenesis and malignant progression, an expression vector harboring human VEGF cDNA was stably transfected into three human cancer cell lines with poor VEGF productivity. Though their in vitro growth rate and intrinsic productivity of another angiogenic factor, basic fibroblast growth factor (bFGF), were not changed by transfection, those clones with higher VEGF production were endowed with tumorigenic and angiogenic potentials as follows: firstly, nontumorigenic, lung carcinoma QG90 cells having lower bFGF productivity acquired tumorigenicity as well as significant in vivo angiogenesis-inducing ability, secondly, tumorigenic colorectal carcinoma RPMI4788 cells having higher potency for bFGF production could form more vascularized solid tumour with faster growth rate and thirdly, oestrogen-dependent breast carcinoma MCF-7 cells, which did not produce detectable bFGF, acquired tumorigenicity even in the absence of oestrogen and the solid tumour growth rate was remarkably enhanced, accompanied with increased vascularization, in the presence of oestrogen. These results suggest that tumour progression closely depends on angiogenesis, and VEGF significantly contributes to malignant progression of a variety of tumour cells through its potent angiogenic activity, independent on the bFGF productivity of tumour cells.     

Endothelial progenitor cell culture for vascular regeneration

A certain population of mononuclear cells in the peripheral blood is capable of contributing to new vessel formation by differentiating into endothelial cells. The basis for native as well as therapeutic neovascularization is not restricted to angiogenesis but includes postnatal vasculogenesis. These cells were discovered by Asahara in 1997 and named endothelial progenitor cells (EPCs). Clinical usefulness of EPCs from human peripheral blood is also suggested in the animal experiments. These results indicate that administering EPCs can be a new clinical strategy for treating ischemic disease, diabetic retinopathy, or neoplasm in which the promotion or inhibition of neovascular formation is critical. However, expansion of EPCs ex vivo is not currently suitable for the clinical setting because of the animal products that are necessary for EPC expansion. To approach the autologous cell-based clinical application of EPCs, we established EPC culture using auto serum. In this article, we will discuss the sources that can generate EPCs and show the usefulness and potential of EPC culture using auto serum in the basic and clinical setting of neovascular formation.