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Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.

Although cardiac stem cells (CSCs) have emerged in regeneration research, the number of isolated CSCs is low, making a sufficient supply of functional elements an important consideration in cardiovascular research. In this study, we established an efficient method for CSC isolation. We directly compared cultures of single cells to human cardiac-derived c-kit-positive progenitor cells (hCPCsc-kit+). The two protocols employed enzymatically digested hCPCsc-kit+ (ED-hCPCs) with tissue-expanded hCPCc-kit+ (TE-hCPCs). Using fluorescence-activated cell sorting, we showed the concentration of c-kit in TE-hCPCs to be higher than in ED-hCPCs, although the total number of c-kit positive cells resulting from ED-hCPCs was similar to that resulting from TE-hCPCs. The cardiomyocyte-associated proteins, GATA4 and Nkx2-5, which were expressed during hCPCs expansion, did not differ between the isolation methods. Importantly, the expression of the CSC stem cell marker, c-kit, was more efficiently preserved using the ED-hCPCs versus the TE-hCPCs method. In a cell proliferation assay, the ED-hCPCs method produced a significantly greater number of cells. Finally, hCPCs derived using both protocols differentiated into endothelial, smooth muscle, and cardiomyocyte lineages. In conclusion, the single-cell culture protocol using an enzymatic digestion method may be more useful to isolate human cardiac-derived c-kit-positive elements compared with the tissue expansion method.

Background Stem cells have a low expansion rate and are difficult to maintain in vitro . To overcome the problems of cardiovascular regeneration, we developed a novel method of stem cell cultivation in culture vessels with amine and carboxyl coatings. Methods and results We isolated cardiac stem/progenitor cells from infant-derived heart tissue by using c-kit antibody (human cardiac-derived c-kit positive progenitor cells; hCPC c-kit+ ); the cells differentiated into endothelial cells, smooth muscle cells, and cardiomyocytes. To characterize the effect of surface modification on hCPC c-kit+ expansion, cellular attachment, c-kit expression maintenance, and cardiomyocyte differentiation, we tested hCPC c-kit+ cultured on non-coated (control), amine-coated (amine), and carboxyl-coated (carboxyl) vessels. Ex vivo proliferation, c-kit maintenance, and cellular attachment were significantly enhanced in the amine group. The amine coating also increased procollagen type I (pro-COL1) expression and increased phosphorylation signals, such as focal adhesion kinase (FAK) and cytosolic Src, as well as enhanced ERK/CDK2 signaling. In addition, there was significant downregulation of the stress signal transducer, JNK, in the amine group. However, cardiomyogenesis remained unchanged in the control, amine, and carboxyl groups. Conclusions Although surface modifications had no effect on early induction cardiomyogenesis, amine-enriched surface modification may increase hCPC c-kit+ expansion. The amine-enriched surface improved cellular proliferation and attachment during ex vivo hCPC c-kit+ expansion, possibly by modulating intracellular signal transducers.

Aims Previous studies on the role of stem cell factor (SCF) in endothelial progenitor cell (EPC)-mediated neovascularization have focused on the EPC mobilization and homing process. However, the direct effects of SCF on neovascularization activity of EPCs have not been characterized. We sought to determine whether SCF regulates the neovascularization ability of EPCs by comparing its roles in mature endothelial cells. Methods and results In vitro and in vivo assays revealed that SCF substantially increased the neovascularization activity of human EPCs through the c-Kit receptor. Notably, the SCF-induced increase in neovascularization activity was substantially greater in EPCs than that in human umbilical vein endothelial cells (HUVECs). SCF-induced phosphorylation of c-Kit and downstream signalling molecules was consistently found to be more potent and longer-lasting in EPCs than in HUVECs. This high responsiveness of EPCs to SCF was explained by the finding that the cell-surface expression of c-Kit is far higher in EPCs than in HUVECs. A c-Kit promoter assay revealed that the increased expression of c-Kit in EPCs could be attributed to the greater expression of stem cell leukaemia, LIM-only 2, and GATA-binding protein 2. Conclusion In addition to its documented role in the mobilization and recruitment of EPCs, our findings show that SCF directly enhances the neovascularization activity of EPCs. Furthermore, the present study provides further evidence that EPCs exhibit differentially greater responsiveness to hypoxia-inducible cytokines, including SCF, than mature endothelial cells, suggesting that EPCs in ischaemic tissues function differently from mature endothelial cells, although they exhibit very similar phenotypes.

The use of cellular therapy in the treatment of dermal wounds is currently an active area of investigation. Multipotent adult stem cells are an attractive choice for cell therapy because they have a large proliferative potential, the ability to differentiate into different cell types and produce a variety cytokines and growth factors important to wound healing. This review focused on the roles of adult stem cells such as endothelial progenitor cells, bone marrow and adipose-derived mesenchymal stem cells, during dermal wound healing process and their therapeutic potentials for the treatment of chronic wounds, which remain a major clinical problem, especially in diabetic patients.

Rationale : Generation of induced pluripotent stem (iPS) cells has been intensively studied by a variety of reprogramming methods, but the molecular and functional properties of the cells differentiated from iPS cells have not been well characterized. Objective : To address this issue, we generated iPS cells from human aortic vascular smooth muscle cells (HASMCs) using lentiviral transduction of defined transcription factors and differentiated these iPS cells back into smooth muscle cells (SMCs). Methods and Results : Established iPS cells were shown to possess properties equivalent to human embryonic stem cells, in terms of the cell surface markers, global mRNA and microRNA expression patterns, epigenetic status of OCT4, REX1, and NANOG promoters, and in vitro/in vivo pluripotency. The cells were differentiated into SMCs to enable a direct, comparative analysis with HASMCs, from which the iPS cells originated. We observed that iPS cell–derived SMCs were very similar to parental HASMCs in gene expression patterns, epigenetic modifications of pluripotency-related genes, and in vitro functional properties. However, the iPS cells still expressed a significant amount of lentiviral transgenes (OCT4 and LIN28) because of partial gene silencing. Conclusions : Our study reports, for the first time, the generation of iPS cells from HASMCs and their differentiation into SMCs. Moreover, a parallel comparative analysis of human iPS cell–derived SMCs and parental HASMCs revealed that iPS-derived cells possessed representative molecular and in vitro functional characteristics of parental HASMCs, suggesting that iPS cells hold great promise as an autologous cell source for patient-specific cell therapy.

Objective: To evaluate the efficacy of the curcumin-coating stent (CCS) on the inhibition of restenosis in a rabbit iliac artery stent model. Results: Curcumin, pigment naturally acquired from the rhizome of the plant curcuma longa, is known to have antiproliferative, antimigratory, and anti-inflammatory effects. However, it is still unclear that curcumin can inhibit neointimal proliferation of the injured vessel. Methods: Dose-dependent inhibition of cell growth was observed over a dose range from 10 nM to 10 μM. CCS was prepared by a dip-coating method (high-dose: HD, low-dose: LD). The release profile of the HD CCS showed that drug release persisted until day 21. Scanning electron microscopy of the CCS showed an intact surface of the stent even after expansion. To test the efficacy of CCS in vivo, LD CCS, HD CCS, and bare metal stents (BMS) were implanted in random order in one iliac artery (N = 30 arteries) of male New Zealand White rabbits (N = 15). Results: After 28 days, the LD and HD CCS groups had a 43% and 55% reduction in the neointimal area, compared with the BMS group (BMS 3.3 ± 1.0 mm2, LD 1.9 ± 0.8 mm2, and HD 0.9 ± 0.5 mm2, P < 0.05). There appeared to be no cytotoxicity related to curcumin at the indicated doses. Conclusions: Curcumin, a natural compound in the human diet, seems to be a safe and effective candidate drug for use in a drug-eluting stent for the prevention of stent restenosis following angioplasty. © 2009 Wiley-Liss, Inc.

ObjectivesThe goal of this study was to investigate the expression of early growth response-1 (Egr-1), a vascular pathogenic transcription factor, and its potential relationship with tissue factor (TF), a key player during the thrombus formation in the abdominal aortic aneurysm (AAA) wall.BackgroundAlthough intraluminal thrombus is a common finding in human AAA, the molecular mechanism of the thrombus formation has not been studied.MethodsDuring the elective AAA repair, specimens were taken from the thrombus-covered and thrombus-free portions of the aneurysmal wall in each of 16 patients with AAA and analyzed to assess the differential expression of Egr-1 and TF. The proinflammatory and prothrombogenic activities of Egr-1 in vasculature were evaluated in vitro and in vivo by overexpressing it using adenovirus.ResultsThe expression of both Egr-1 and TF was significantly increased in the thrombus-covered wall compared with the thrombus-free wall, in which their up-regulation in the thrombus-covered wall was strongly correlated with each other (p < 0.005, r = 0.717). Adenoviral overexpression of Egr-1 in human vascular smooth muscle and endothelial cells was found to up-regulate the expression of TF and inflammation-related genes. Moreover, Egr-1 overexpression in endothelial cells increased their adhesiveness to monocytes and also substantially promoted the intravascular thrombus formation in vivo, as shown in the inferior vena cava ligation experiment of the rat.ConclusionsThe present study demonstrates the differential up-regulation of Egr-1 in the thrombus-covered wall of human AAA and also suggests its possible contribution to the thrombogenic and inflammatory pathogenesis in human AAA.

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures1. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system2,3; however, their role in the mammalian vasculature remains ill defined4–8. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165–induced migration, tube formation and permeability in vitro and VEGF-165–stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.

Few studies have examined in detail the combined effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene delivery on collateral development. Here, we evaluated the potential synergism of naked DNA vectors encoding VEGF and bFGF using a skeletal-muscle based ex vivo angiogenesis assay and compared tissue perfusion and limb loss in a murine model of hindlimb ischemia. In the ex vivo angiogenesis assay, the VEGF+bFGF combination group had a larger capillary sprouting area than those of the LacZ, VEGF, and bFGF groups. Consistent with these results, regional blood flow recovery on day 14 was also highest in the VEGF+bFGF combination group, followed by the bFGF, VEGF, and LacZ groups. The limb loss frequency was 0% in the combination group, whereas the limb loss frequencies of the other groups were 7-29%. The ischemic muscles of the combination group revealed evidence of increased angiogenesis and arteriogenesis and the upregulated expression of genes that may be associated with arteriogenesis, such as those for cardiac ankyrin repeat protein, early growth response factor-1, and transforming growth factor-beta1. Our study has implications for the development of a combined gene therapy for the vascular occlusive diseases.

Objective: Ischemia-dependent upregulation of angiopoietin2 (Ang2) led us to hypothesize the potentially proangiogenic Ang2-Tie2 signaling in endothelial progenitor cells (EPCs). Given the well-known vascular destabilizing action of Ang2 in mature endothelium, we investigated the yet unidentified mechanism behind cell-dependent differential activity of Ang2. Methods and results: Both in vitro and in vivo experiments showed that Ang2 promoted angiogenicity of human cord blood-derived EPCs, where Ang2 directly activated Tie2 and its related downstream signaling molecules. However, Ang2 had no such effect in fully differentiated human umbilical vein endothelial cells (HUVECs) under the same condition. Such a cell-dependent Tie2 activation by Ang2 was explained by comparing EPCs and HUVECs, where most Tie2 receptors in EPCs were found to be present unbound to Tie1, whereas those in HUVECs existed as heterocomplexes with Tie1. When Tie2 in HUVECs was prevented from forming heterocomplexes by silencing Tie1 expression, they underwent rapid phosphorylation upon Ang2 treatment, as shown in EPCs. Conclusions: In contrast with its roles in mature endothelial cells, Ang2 has proangiogenic activities in EPC directly through Tie2 signaling pathway. Such a cell-dependent differential reactivity of Ang2 was for the first time found to be modulated by physical association between Tie1 and Tie2, which inhibited Ang2-mediated Tie2 activation.

Endothelial progenitor cells (EPCs) act as endothelial precursors that promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. These facts prompt the hypothesis that EPC transplantation should accelerate the wound-repair process by facilitating neovascularization and the production of various molecules related to wound healing. In a murine dermal excisional wound model, EPC transplantation accelerated wound re-epithelialization compared with the transplantation of mature endothelial cells (ECs) in control mice. When the wounds were analyzed immunohistochemically, the EPC-transplanted group exhibited significantly more monocytes/macrophages in the wound at day 5 after injury than did the EC-transplanted group. This observation is consistent with enzyme-linked immunosorbent assay results showing that EPCs produced in abundance several chemoattractants of monocytes and macrophages that are known to play a pivotal role in the early phase of wound healing. At day 14 after injury, the EPC-transplanted group showed a statistically significant increase in vascular density in the granulation tissue relative to that of the EC-transplanted group. Fluorescence microscopy revealed that EPCs preferentially moved into the wound and were directly incorporated into newly formed capillaries in the granulation tissue. These results suggest that EPC transplantation will be useful in dermal wound repair and skin regeneration, because EPCs both promote the recruitment of monocytes/macrophages into the wound and increase neovascularization.

The effect of aldosterone on connective tissue growth factor (CTGF) was examined in rat embryonic ventricular myocytes. Upon aldosterone treatment, CTGF expression was significantly increased in a dose and time-dependent manner. To explore the molecular mechanism for this upregulation, we examined the role of mineralocorticoid receptor. Pre-treatment of an antagonist (spironolactone) at 5-fold excess of aldosterone blocked the CTGF induction by aldosterone, suggesting that the upregulation was mediated by mineralocorticoid receptor. Aldosterone treatment resulted in activation of ERK1/2, p38 MAPK, and JNK pathways with a more transient pattern in p38 MAPK. Blocking studies using pre-treatment of the inhibitor of each pathway revealed that p38 MAPK cascade may be important for aldosterone-mediated CTGF upregulation as evidenced by the blocking of CTGF induction by SB203580 (p38 MAPK inhibitor), but not by PD098059 (ERK1/2 inhibitor) and JNK inhibitor I. Interestingly, JNK inhibitor I and PD098059 decreased the basal level of CTGF expression. On the other hand, pre-treatment of spironolactone abrogated the p38 MAPK activation, indicating that mineralocorticoid receptor mechanism is linked to p38 MAPK pathway. Taken together, our findings suggest that aldosterone induces CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor and that cross-talk exists between the two pathways.

C-reactive protein (CRP), a predictor of future cardiovascular diseases, has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation. This proatherogenic CRP was speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells (EPCs), possibly impairing vascular regeneration and increasing cardiovascular vulnerability to ischemic injury. Herein, we investigated the direct effect of CRP on angiogenic activity and gene expression in human EPCs. Incubation of EPCs with human recombinant CRP significantly inhibited EPC migration in response to vascular endothelial growth factor, possibly by decreasing the expression of endothelial nitric oxide synthase and subsequent nitric oxide production. In addition, CRP-treated EPCs showed the reduced adhesiveness onto an endothelial cell monolayer. When assayed for the gene expression of arteriogenic chemo-cytokines, CRP substantially decreased their expression levels in EPC, in part due to the upregulation of suppressors of cytokine signaling proteins. These results suggest that CRP directly attenuates the angiogenic and possibly arteriogenic functions of EPCs. This CRP-induced EPC dysfunction may impair the vascular regenerative capacity of EPCs, thereby leading to increased risk of cardiovascular diseases.

Objective— Increased risk of cardiovascular disease in patients with chronic renal failure (CRF) has been explained by accelerated atherosclerosis and impaired angiogenesis, in which endothelial progenitor cells (EPCs) may play key roles. We hypothesized that altered EPC biology may contribute to the pathophysiology of CRF. Methods and Results— EPCs were isolated from CRF patients on maintenance hemodialysis (n=44) and from a normal control group (n=30). CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) when compared with the controls ( P P =0.040) and 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC) ( P r =−0.461, P =0.010) and normal group ( r =−0.367, P =0.016) significantly correlated with the numbers of EPC. Indeed, the number of circulating EPC was significantly lower in CRF patients than in normal group under the same burden of risk factors ( P r =0.427, P =0.004). Conclusions— EPC biology, which is critical for neovascularization and the maintenance of vascular function, is altered in CRF. Our data strongly suggest that dysfunction of circulating EPC has a role in the progression of cardiovascular disease in patients with CRF.

To test the hypothesis that overexpression of early growth response factor-1 (Egr-1) contributes to the revascularization of ischemic limbs, a constitutively active form of Egr-1 (Egr-1*) was made and evaluated in vitro and in vivo. Analyses of the transduced myocytes revealed significant upregulation of bFGF, PDGF-A, PDGF-B, IGF-II, and TGF-beta1. A coculture assay of the paracrine effects indicated that Ad-Egr-1* promoted proliferation and migration of endothelial cells. When Ad-Egr-1* was injected into the tibialis anterior muscle of mice, followed by explant culture in growth factor-reduced Matrigel, many capillary-like structures were observed in the Egr-1* group compared with minimal sprouting from the LacZ group, suggesting an angiogenic potential of Egr-1*. Next we evaluated Ad-Egr-1* in a murine model of hindlimb ischemia. Compared with slow revascularization in the control PBS or LacZ group, a rapid increase in tissue perfusion was observed in the Egr-1* group and the difference in flux ratio was statistically significant at day 7. In the injected muscle, expression of Egr-1*, upregulation of its target genes, and increased number of vessels staining positive for smooth muscle alpha-actin were observed. These results suggest that Egr-1 plays an important role in vascular recovery after occlusion and could be a potential target for therapeutic angiogenesis.

An angiogenesis assay based on gene transfer would be extremely useful for angiogenic gene therapy. A simple, reproducible, and quantitative assay to test angiogenic genes would provide more accurate predictions than conventional peptide-based assays. Here, we have developed a semiquantitative angiogenesis assay utilizing gene transfer into skeletal muscle, which is a target tissue for ischemic limb diseases. To facilitate quick and clean analysis, a naked plasmid DNA vector combined with an electroporation procedure was used for gene transfer. When the plasmid vector encoding vascular endothelial growth factor cDNA (pJDK-VEGF165) was injected into the tibialis anterior muscle of BALB/c mice, followed by in vivo electroporation and explant culture in growth factor-reduced Matrigel, the outward migration of sprouting cells was observed as early as day 2. The cells soon formed capillary networks, which peaked at day 7 and persisted until day 14. The capillary-like structures were positive for von Willebrand factor, platelet endothelial cell adhesion molecule, and vimentin, suggesting they were endothelial cells. There was little, if any, sprouting or formation of capillaries from the control vector (pJDK)-injected group. Consistent with the region of sprouting and network formation, the amount of secreted VEGF increased in the conditioned medium of explant cultures. The angiogenic potential of connective tissue growth factor (CTGF) was examined using the new assay. Whereas the CTGF gene alone induced weak sprouting activity, it appeared to inhibit the angiogenic activity of the VEGF165 gene during cotreatment. This attenuating activity of CTGF on VEGF was reproduced in vivo in a murine model of hindlimb ischemia. In a group of mice treated with both pJDK-CTGF and pJDK-VEGF165, the blood flow measured by laser Doppler imaging was significantly lower than that of the pJDK-VEGF165-treated group 10 days after femoral artery excision. These results are consistent with recent reports that suggest that CTGF inhibits VEGF. This confirms the usefulness of this novel ex vivo assay in assessing the angiogenic capacity of genes of interest. In summary, this new gene-based angiogenesis assay should be widely applicable in the study of angiogenic or antiangiogenic genes because it can readily predict the angiogenic potential of specific genes and their combinations.

We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and α-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both α-SMA and SM22-α proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.

Nat. Med. 14, 448–453 (2008); published online 16 March 2008; corrected after print 18 April 2008 In the version of this article initially published, the affiliation of Rebecca A. Stockton was incorrect. Her correct affiliation is affiliation 5: the Department of Medicine, University of California, San Diego, 9500 Gilman Dr.

To develop a potent hypoxia-inducible vector, we evaluated the usefulness of chimeric combinations of the early growth response factor-1 (Egr-1)-binding site (EBS) from the Egr-1 promoter, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 (PGK1) gene. In transient trasfection assays, combination of three copies of HRE (3xHRE) with either EBS or MRE resulted in a significant increase in hypoxia-responsiveness. With three-enhancer combination such as the EBS-MRE-3xHRE (E-M-H), a hypoxia induction ratio of 69 was achieved. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus (HCMV) promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. The E-M-H was also tested for induction by hypoxia mimetics and other stresses such as heat, low glucose, low pH, and hydrogen peroxide. Co2+, deferoxamine, and Ni2+turned out to be potent inducers whereas hydrogen peroxide worked as a moderate inducer. Gel shift assay together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1|[alpha]| (HIF-1|[alpha]|), metal transcription factor-1 (MTF-1), and Egr-1 may be associated with the high inducibility of E-M-H enhancer. In vivo evaluation of the E-M-H vector in ischemic calf muscle revealed that luciferase expression was significantly higher in E-M-H group than in control, H, or HCMV group. With its high induction capacity and versatile means of modulation, the novel chimeric enhancer should find wide application in the treatment of ischemic diseases and cancer.

Top of pageAbstract We hypothesized that the overexpression of early growth response factor-1 (Egr-1), which plays a pivotal role in the activation of many genes under stress conditions, might induce multiple genes involved in vessel formation and thereby contribute to the revascularization of ischemic muscle. To test this hypothesis, adenoviral vectors carrying the genes for Egr-1 or Egr-1|[ast]| (a constitutively active form of Egr-1) were made and transduced into primary human skeletal myocytes. RNA and protein analyses revealed significant upregulation of angiogenesis-related genes, such as bFGF, PDGF-A, PDGF-B, IGF-II, and TGF-|[beta]|1. A co-culture assay of the paracrine effects of transduced myocytes indicated that the Ad-Egr-1|[ast]| group significantly promoted proliferation of endothelial cells and regrowth from the wound edge following scratch injury. To further evaluate the angiogenic potential of Egr-1|[ast]|, Ad-Egr-1|[ast]| was injected into the tibialis anterior muscle of mouse (Balb/c), followed by explant culture in growth factor-reduced Matrigel. Many sprouted capillary-like structures were observed in the Egr-1|[ast]| group compared with minimal sprouting from the LacZ group, suggesting an angiogenic activity of Egr-1|[ast]|. Next we evaluated Ad-Egr-1|[ast]| in vivo in a murine model of hindlimb ischemia (Balb/c). Compared with the slow revascularization in the control group (PBS or Ad-LacZ), a rapid increase in tissue perfusion was observed in the Ad-Egr-1|[ast]| group and the difference in the measured flux ratio was statistically significant at day 7. Expression of Egr-1|[ast]|, upregulation of its target genes, and increased number of vessels that stained positive for smooth muscle |[alpha]|-actin (|[alpha]|-SMA) were also observed in the injected muscle. These results suggest that Egr-1 plays an important role in vascular recovery after occlusion and could be a potential target for therapeutic angiogenesis.

Objective—Increased risk of cardiovascular disease in patients with chronic renal failure (CRF) has been explained by accelerated atherosclerosis and impaired angiogenesis, in which endothelial progenitor cells (EPCs) may play key roles. We hypothesized that altered EPC biology may contribute to the pathophysiology of CRF. Methods and Results—EPCs were isolated from CRF patients on maintenance hemodialysis (n44) and from a normal control group (n30). CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) when compared with the controls (P0.001). These findings were corroborated by 30.5% decrease in EPC migratory function in response to vascular endothelial growth factor (VEGF) (P0.040) and 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC) (P0.001). In addition, Framingham’s risk factor score of both CRF (r0.461, P0.010) and normal group (r0.367, P0.016) significantly correlated with the numbers of EPC. Indeed, the number of circulating EPC was significantly lower in CRF patients than in normal group under the same burden of risk factors (P0.001). A significant correlation was also observed between dialysis dose (Kt/V) and EPC incorporation into HUVEC (r0.427, P0.004). Conclusions—EPC biology, which is critical for neovascularization and the maintenance of vascular function, is altered in CRF. Our data strongly suggest that dysfunction of circulating EPC has a role in the progression of cardiovascular disease in patients with CRF. (Arterioscler Thromb Vasc Biol. 2004;24:1246-1252.)