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Human induced pluripotent stem cells (hiPSCs) hold great promise for cardiovascular regeneration following ischemic injury. Considerable effort has been made toward the development and optimization of methods to differentiate hiPSCs into vascular cells, such as endothelial and smooth muscle cells (ECs and SMCs). In particular, hiPSC-derived ECs have shown robust potential for promoting neovascularization in animal models of cardiovascular diseases, potentially achieving significant and sustained therapeutic benefits. However, the use of hiPSC-derived SMCs that possess high therapeutic relevance is a relatively new area of investigation, still in the earlier investigational stages. In this review, we first discuss different methodologies to derive vascular cells from hiPSCs with a particular emphasis on the role of key developmental signals. Furthermore, we propose a standardized framework for assessing and defining the EC and SMC identity that might be suitable for inducing tissue repair and regeneration. We then highlight the regenerative effects of hiPSC-derived vascular cells on animal models of myocardial infarction and hindlimb ischemia. Finally, we address several obstacles that need to be overcome to fully implement the use of hiPSC-derived vascular cells for clinical application.

Preeclampsia (PE) is a prevalent pregnancy disorder that leads to high maternal and fetal morbidity and mortality. While defective vascular development and angiogenesis in placenta are known as crucial pathological findings, its pathophysiological mechanism remains elusive. To better understand the effects of PE on angio-vasculogenesis and inflammatory networks in the fetus and to identify their biological signatures, we investigated the quantitative and functional characteristics of cord blood-derived mononuclear cells (CB-MNCs) and CD31-positive MNCs. Flow cytometry analysis demonstrated that the CB-MNCs from the severe PE group had significantly decreased number of cells expressing CD3, CD11b, CD14, CD19, KDR, and CD31 compared with the normal group. Quantitative real time PCR (qRT-PCR) shows down-regulation of the major angiogenic factor VEGFA in MNCs and CD31+ MNCs in severe PE. The major inflammatory cytokines IL1 was highly upregulated in CD31+ CB-MNCs in the severe PE patients. Mild PE patients, however, did not display any significant difference in expression of all measured angiogenic genes and most inflammatory genes. These findings show distinct angiogenic and inflammatory signatures from severe PE, and they may play a significant role in the pathogenesis of vascular defects in placenta of severe PE.

Atherosclerosis is a chronic inflammatory disease driven by lipid accumulation in arteries, leading to narrowing and thrombosis. It affects the heart, brain, and peripheral vessels and is the leading cause of mortality in the United States. Researchers have strived to design nanomaterials of various functions, ranging from non-invasive imaging contrast agents, targeted therapeutic delivery systems to multifunctional nanoagents able to target, diagnose, and treat atherosclerosis. Therefore, this review aims to summarize recent progress (2017-now) in the development of nanomaterials and their applications to improve atherosclerosis diagnosis and therapy during the preclinical and clinical stages of the disease.

Transplantations of various stem cells or their progeny have repeatedly improved cardiac performance in animal models of myocardial injury; however, the benefits observed in clinical trials have been generally less consistent. Some of the recognized challenges are poor engraftment of implanted cells and, in the case of human cardiomyocytes, functional immaturity and lack of electrical integration, leading to limited contribution to the heart’s contractile activity and increased arrhythmogenic risks. Advances in tissue and genetic engineering techniques are expected to improve the survival and integration of transplanted cells, and to support structural, functional, and bioenergetic recovery of the recipient hearts. Specifically, application of a prefabricated cardiac tissue patch to prevent dilation and to improve pumping efficiency of the infarcted heart offers a promising strategy for making stem cell therapy a clinical reality.

Cardiovascular disease is presently the number one cause of death worldwide. Current stents used to treat cardiovascular disease have a litany of unacceptable shortcomings: adverse clinical events including restenosis, neointimal hyperplasia, thrombosis, inflammation, and poor re-endothelialization. We have developed a biocompatible, multifunctional, peptide amphiphile-based nanomatrix coating for stents. In this study, we evaluated the ability of the nanomatrix coated stent to simultaneously address the issues facing current stents under physiological flow conditions in vitro. We found that the nanomatrix coated stent could increase endothelial cell migration, adhesion, and proliferation (potential for re-endothelialization), discourage smooth muscle cell migration and adhesion (potential to reduce neointimal hyperplasia and restenosis), and decrease both platelet activation and adhesion (potential to prevent thrombosis) as well as monocyte adhesion (potential to attenuate inflammatory responses) under physiological flow conditions in vitro. These promising results demonstrate the potential clinical utility of this nanomatrix stent coating, and highlight the importance of biocompatibility, multifunctionality, and bioactivity in cardiovascular device design.

Rationale: For therapeutic neovascularization through cell therapy to treat cardiovascular diseases, recently, we successfully generated reprogrammed ECs, directly from human dermal fibroblasts (HDF) with the ETV2 transcription factor. Although functional ECs were generated, ETV2 was delivered lentiviral vector, which is clinically unsuitable. Objective: In this study, we aimed to generate clinically compatible rECs, by employing an adenoviral vector to minimize genetic integration. Furthermore, to prolong the cell retention and improve cell survival, we applied synthetic biomaterial, polypeptide amphiphile (PA) nanomatrix gel and tested for experimental cell therapy in animal models. Methods and Results: After we transduced HDFs with Ad- ETV2 (Ad- ETV2 -HDF), EC gene expression was measured by qRT-PCR and flow cytometry. We found that Ad- ETV2- HDFs displayed a cobblestone-like morphology and showed notable induction of EC genes (CDH5, KDR, PECAM1). Flow cytometry analysis showed peak expression of CDH5 and KDR at D6 and continuous increase of PECAM1 over 9 days. Also, Ad- ETV2 -HDFs took up acetylated LDL and bound lectin, suggesting that these cells possess functional EC characteristics. To enrich cells showing EC characters, Ad- ETV 2-HDFs were sorted for KDR by FACS at day 6. qRT-PCR showed substantial enrichment of EC-specific gene expression in KDR + cells. Only KDR + cells (reprogrammed ECs, rECs) formed tube-like structures on Matrigel. In addition, immunostaining confirmed high expression of EC marker proteins in rECs. Next, we tested the functionality of rECs in hindlimb ischemia mouse model. At 4 weeks after injection of 4 x 10 5 rECs into the ischemic hindlimb, we observed rECs incorporated into host blood vessels or resided perivascular regions. Furthermore, we found more rECs in the tissues and were directly incorporated into the vessels when they were encapsulated in PA nanomatrix gel compared to bare rEC-injected mice. Conclusions: Together, these data strongly support the generation of Ad- ETV2 -induced rECs (Ad-rECs) and enhanced retention of rECs by PA nanomatrix gel in tissue. With this study, we can develop advanced concept and techniques for clinical application of rECs and establish a new platform for future cell therapy.

Attempts to regenerate cardiac vessels in order to treat ischemic myocardial disease have continued to be an old goal in the field of gene therapy. However, most studies that have applied proangiogenic factors have shown that the effects were limited. We examined the therapeutic effects of the ETV2 gene, a factor induced by postnatal injury, which is involved in the differentiation and specification of endothelial cells in the developmental stages in animal models of ischemic myocardial infarction via the adenoviral expression system. The ETV2 gene, using adenoviral vectors, was efficiently delivered in normal heart and myocardial infarction models and induced gene expression. In the myocardial infarction model of the control group, the function of the heart deteriorated after induction over time, but deterioration in the adenoviral ETV2-treated myocardial infarction group was delayed. To confirm whether this effect was due to angiogenesis, the expression of proangiogenic factors was examined, and the expression of vascular endothelial growth factor and angiopoietin were significantly increased in the group treated with ETV2. This result was reproduced via ETV2 gene expression at the cellular level. In order to confirm the effects of angiogenesis more deeply, we confirmed that the dividing vascular endothelial cells expressed Ki67. As a result, it was confirmed that a significant number of Ki67-positive cells were present in the functional blood vessel region in the group treated with ETV2. Taken together, the results suggest that angiogenesis plays a role in regenerating cardiac vessels when the ETV2 gene is introduced into the myocardial infarction model, which may be considered as a candidate for treatment of ischemic heart disease.

Recent evidence has suggested that diabetic neuropathy (DN) is pathophysiologically related to both impaired angiogenesis and a deficiency of neurotrophic factors in the nerves. It is widely known that vascular and neural growths are intimately associated. Mesenchymal stem cells (MSCs) promote angiogenesis in ischemic diseases and have neuroprotective effects, particularly on Schwann cells. Accordingly, we investigated whether DN could be improved by local transplantation of MSCs by augmenting angiogenesis and neural regeneration such as remyelination. In sciatic nerves of streptozotocin (STZ)-induced diabetic rats, motor and sensory nerve conduction velocities (NCVs) and capillary density were reduced, and axonal atrophy and demyelination were observed. After injection of bone marrow-derived MSCs (BM-MSCs) into hindlimb muscles, NCVs were restored to near-normal levels. Histological examination demonstrated that injected MSCs were preferentially and durably engrafted in the sciatic nerves, and a portion of the engrafted MSCs were distinctively localized close to vasa nervora of sciatic nerves. Furthermore, vasa nervora increased in density, and the ultrastructure of myelinated fibers in nerves was observed to be restored. Real-time RT-PCR experiments showed that gene expression of multiple factors involved in angiogenesis, neural function, and myelination were increased in the MSC-injected nerves. These findings suggest that MSC transplantation improved DN through direct peripheral nerve angiogenesis, neurotrophic effects, and restoration of myelination.

Background This study sought to systematically investigate the derivation of late outgrowth endothelial progenitor cells (late EPC) and mesenchymal stem cells (MSC) from umbilical cord blood (UCB) and to examine their therapeutic effects on myocardial infarction (MI). Methods The expression of angiogenic genes was determined by qRT-PCR. Myocardial infarction (MI) was induced in rats, and cells were directly transplanted into the border regions of ischemic heart tissue. Results Culture of UCB mononuclear cells yielded two distinct types of cells by morphology after 2weeks in the same culture conditions. These cells were identified as late EPC and MSC, and each was intramyocardially injected into rat hearts after induction of MI. Echocardiography and histologic analyses demonstrated that both EPC and MSC improved cardiac function and enhanced vascularization, although fibrosis was reduced only in the EPC transplanted hearts. Different paracrine factors were enriched in EPC and MSC. However, once injected into the hearts, they induced similar types of paracrine factors in the heart. Transplanted EPC or MSC were mostly localized at the perivascular areas. This study demonstrated that EPC and MSC can be simultaneously derived from UCB under the same initial culture conditions, and that common paracrine factors are involved in the repair of MI. Conclusion Late EPC and MSC are effective for infarct repair, apparently mediated through common humoral mechanisms.

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) emerged as an important source of cells for cardiovascular regeneration. This review summarizes protocols for generating hPSC-ECs and provides an overview of the current state of the research in clinical application of hPSC-derived ECs. Various systems were developed for differentiating hPSCs into the EC lineage. Stepwise two-dimensional systems are now well established, in which various growth factors, small molecules, and coating materials are used at specific developmental stages. Moreover, studies made significant advances in clinical applicability of hPSC-ECs by removing undefined components from the differentiation system, improving the differentiation efficiency, and proving their direct vascular incorporating effects, which contrast with adult stem cells and their therapeutic effects in vivo. Finally, by using biomaterial-mediated delivery, investigators improved the survival of hPSC-ECs to more than 10 months in ischemic tissues and described long-term behavior and safety of in vivo transplanted hPSC-ECs at the histological level. hPSC-derived ECs can be as a critical source of cells for treating advanced cardiovascular diseases. Over the past two decades, substantial improvement has been made in the differentiation systems and their clinical compatibility. In the near future, establishment of fully defined differentiation systems and proof of the advantages of biomaterial-mediated cell delivery, with some additional pre-clinical studies, will move this therapy into a vital option for treating those diseases that cannot be managed by currently available therapies.

The purpose of this study was to investigate the effects of diabetes on mesenchymal stem cells (MSCs) in terms of their angiogenic and therapeutic potential for repairing tissue ischemia. We culture-isolated MSCs from streptozotocin-induced diabetic rats (D-MSCs) and compared their proliferation, differentiation, and angiogenic effects with those from normal rats (N-MSCs). The angiogenic effects of MSCs were evaluated by real-time PCR, in vitro tube formation assay, and transplantation of the MSCs into a hindlimb ischemia model followed by laser Doppler perfusion imaging. The number of MSCs derived from diabetic rats was smaller, and their proliferation rate was slower than N-MSCs. Upon induction of differentiation, the osteogenic and angiogenic differentiation of D-MSCs were aberrant compared to N-MSCs. The expression of angiogenic factors was lower in D-MSCs than N-MSCs. D-MSCs cocultured with endothelial cells resulted in decreased tube formation compared to N-MSCs. D-MSCs were ineffective to improve hindlimb ischemia and showed lower capillary density and angiogenic gene expression in ischemic limbs than N-MSCs. D-MSCs have defective proliferation and angiogenic activities and are ineffective for repairing hindlimb ischemia. Newer measures are needed before MSCs can be employed as a source for autologous cell therapy.

BackgroundCell therapy for cardiovascular disease has been limited by low engraftment of administered cells and modest therapeutic effects. Bone marrow (BM) -derived CD31+ cells are a promising cell source owing to their high angiovasculogenic and paracrine activities.ObjectivesThis study sought to identify culture conditions that could augment the cell adhesion, angiogenic, and anti-inflammatory activities of BM-derived CD31+ cells, and to determine whether these cultured CD31+ cells are effective for cardiac and vascular repair.MethodsCD31+ cells were isolated from human BM by magnetic-activated cell sorting and cultured for 10 days under hematopoietic stem cell, mesenchymal stem cell, or endothelial cell culture conditions. These cells were characterized by adhesion, angiogenesis, and inflammatory assays. The best of the cultured cells were implanted into myocardial infarction (MI) and hindlimb ischemia (HLI) models to determine therapeutic effects and underlying mechanisms.ResultsThe CD31+ cells cultured in endothelial cell medium (EC-CD31+ cells) showed the highest adhesion and angiogenic activities and lowest inflammatory properties in vitro compared with uncultured or other cultured CD31+ cells. When implanted into mouse MI or HLI models, EC-CD31+ cells improved cardiac function and repaired limb ischemia to a greater extent than uncultured CD31+ cells. Histologically, injected EC-CD31+ cells exhibited higher retention, neovascularization, and cardiomyocyte proliferation. Importantly, cell retention and endothelial transdifferentiation was sustained up to 1 year.ConclusionsShort-term cultured EC-CD31+ cells have higher cell engraftment, vessel-formation, cardiomyocyte proliferation, and anti-inflammatory potential, are highly effective for both cardiac and peripheral vascular repair, and enhance survival of mice with heart failure. These cultured CD31+ cells may be a promising source for treating ischemic cardiovascular diseases.

Background Postsurgical secondary lymphedema is usually a progressive and lifelong condition lacking any curative treatment. The aim of this study was to develop new, simple surgical mouse models of chronic lymphedema, better simulating chronic nature of human postsurgical lymphedema. Methods Two experimental mouse models of secondary lymphedema were created surgically without radiation by modifications of the previously described methods: the tail model and the hind limb model. Lymphedema formation was clinically assessed and quantitatively evaluated by measuring circumferences and limb volumes. Postmortem specimens were assessed histologically to examine the efficacy of the models. Results In the tail models, although a substantial frequency of tail necrosis (30.0%) was noted and the increase in circumference was maintained for only limited times postoperatively depending on the particular tail model, the overall success rate was 65.0%. In the mouse hind limb model, the overall success rate was 88.9%, and the increased circumference and limb volume were maintained over the entire study period of 8 weeks. The overall success rate of the mouse hind limb model was significantly higher than that of the mouse tail model(s). Conclusions We have successfully established modified mouse tail and hind limb lymphedema models via only surgical techniques without radiation, which have characteristics of chronic secondary lymphedema. The mouse hind limb model has a higher success rate than the mouse tail model and has advantages of having the healthy contralateral hind limbs as an internal control.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which are collectively called pluripotent stem cells (PSCs), have emerged as a promising source for regenerative medicine. Particularly, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have shown robust potential for regenerating injured heart. Over the past two decades, protocols to differentiate hPSCs into CMs at high efficiency have been developed, opening the door for clinical application. Studies further demonstrated therapeutic effects of hPSC-CMs in small and large animal models and the underlying mechanisms of cardiac repair. However, gaps remain in explanations of the therapeutic effects of engrafted hPSC-CMs. In addition, bioengineering technologies improved survival and therapeutic effects of hPSC-CMs in vivo. While most of the original concerns associated with the use of hPSCs have been addressed, several issues remain to be resolved such as immaturity of transplanted cells, lack of electrical integration leading to arrhythmogenic risk, and tumorigenicity. Cell therapy with hPSC-CMs has shown great potential for biological therapy of injured heart; however, more studies are needed to ensure the therapeutic effects, underlying mechanisms, and safety, before this technology can be applied clinically.

Delayed reendothelialization contributes to restenosis after angioplasty and stenting in diabetes. Prior data have shown that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to endothelial recovery after arterial injury. We investigated the hypothesis that the EPC contribution to reendothelialization may be impaired in diabetes, resulting in delayed reendothelialization. Reendothelialization was significantly reduced in diabetic mice compared with nondiabetic mice in a wire-induced carotid denudation model. The EPC contribution to neoendothelium was significantly reduced in Tie2/LacZ BM-transplanted diabetic versus nondiabetic mice. BM from diabetic and nondiabetic mice was transplanted into nondiabetic mice, revealing that reendothelialization was impaired in the recipients of diabetic BM. To examine the relative roles of denuded artery versus EPCs in diabetes, we injected diabetic and nondiabetic EPCs intravenously after arterial injury in diabetic and nondiabetic mice. Diabetic EPCs recruitment to the neoendothelium was significantly reduced, regardless of the diabetic status of the recipient mice. In vitro, diabetic EPCs exhibited decreased migration and adhesion activities. Vascular endothelial growth factor and endothelial NO synthase expressions were also significantly reduced in diabetic EPCs. Notably, thrombospondin-1 mRNA expression was significantly upregulated in diabetic EPCs, associating with the decreased EPC adhesion activity in vitro and in vivo. Reendothelialization is impaired by malfunctioning EPCs in diabetes. Diabetic EPCs have phenotypic differences involving thrombospondin-1 expression compared with nondiabetic EPCs, revealing potential novel mechanistic insights and therapeutic targets to improve reendothelialization and reduce restenosis in diabetes.

Background— Peripheral neuropathy is a frequent and major complication of diabetes. Methods and Results— Severe peripheral neuropathy developed in type II diabetic mice, characterized by significant slowing of motor and sensory nerve conduction velocities. Rosuvastatin restored nerve vascularity, including vessel size, and nerve function also recovered to the levels of nondiabetic mice. Neuronal nitric oxide synthase expression in sciatic nerves was reduced in diabetic mice but was preserved by rosuvastatin. Coadministration of a nitric oxide synthase inhibitor with rosuvastatin attenuated the beneficial effects of rosuvastatin on nerve function and limited the recovery of vasa nervorum and nerve function. In vitro, rosuvastatin inhibited downregulation of neuronal nitric oxide synthase expression induced by high-glucose conditions in cultured Schwann cells. Furthermore, Akt phosphorylation in Schwann cells, downregulated by high-glucose conditions, was also restored by rosuvastatin, consistent with the change of neuronal nitric oxide synthase expression. Akt inhibition independently reduced neuronal nitric oxide synthase expression in Schwann cells in low-glucose cultures. Conclusions— These data indicate that the HMG-CoA reductase inhibitor rosuvastatin has a favorable effect on diabetic neuropathy independent of its cholesterol-lowering effect. Our data provide evidence that this effect may be mediated in part via neuronal nitric oxide synthase/nitric oxide and phosphatidylinositol 3-kinase/Akt-signaling pathways and also suggest that restoration or preservation of the microcirculation of the sciatic nerve may be involved.

Background— Toxic neuropathies induced by cisplatin and other chemotherapeutic agents are important clinical problems because of their high incidence, their lack of effective treatment, and the fact that neuropathy represents a dose-limiting factor for these therapies. The pathogenic basis for toxic neuropathies induced by chemotherapeutic agents has not been completely elucidated. Methods and Results— We investigated the hypothesis that experimental toxic neuropathy results from an antiangiogenic effect of these drugs, resulting in destruction of the vasa nervorum, and accordingly that the neuropathy could be prevented or reversed by locally administered VEGF gene transfer without augmenting tumor growth. In an animal model of cisplatin-induced neuropathy, nerve blood flow was markedly attenuated, and there was a profound reduction in the number of vasa nervorum associated with marked endothelial cell apoptosis, resulting in a severe peripheral neuropathy with focal axonal degeneration characteristic of ischemic neuropathy. After intramuscular gene transfer of naked plasmid DNA encoding VEGF-1 in animals with an established neuropathy, vascularity and blood flow returned to levels similar to those of control rats, peripheral nerve function was restored, and histological nerve architecture was normalized. Gene therapy administered in parallel with cisplatin chemotherapy completely attenuated endothelial cell apoptosis and inhibited destruction of nerve vasculature, deterioration of nerve function, and axonal degeneration. In a rat tumor model, VEGF gene transfer administered locally did not alter tumor growth or vascularity. Conclusions— These findings implicate microvascular damage as the basis for toxic neuropathy induced by cisplatin and suggest that local angiogenic gene therapy may constitute a novel prevention or treatment for this disorder without augmenting tumor growth or vascularization.

Background— Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis, which leads to progressive heart failure; however, the pathogenesis of DCM remains uncertain. Methods and Results— Using the streptozotocin-induced diabetic rat model, we evaluated the natural course of DCM over a period of 1 year by serial echocardiography, Western blot analysis for vascular endothelial growth factor (VEGF), endothelial progenitor cell assays, myocardial blood flow measurements, and histopathologic analysis that included terminal dUTP nick end-labeling (TUNEL), capillary and cardiomyocyte density, and fibrosis area. Downregulation of myocardial VEGF expression preceded all other features of DCM and was followed by increased apoptosis of endothelial cells, decreased numbers of circulating endothelial progenitor cells, decreased capillary density, and impaired myocardial perfusion. Apoptosis and necrosis of cardiomyocytes ensued, along with fibrosis and progressive diastolic and then systolic dysfunction. To provide further evidence of the central role of VEGF in the pathophysiology of DCM, we replenished myocardial VEGF expression using naked DNA gene therapy via direct intramyocardial injection of plasmid DNA encoding VEGF (phVEGF 165 ). VEGF-replenished rats showed increased capillary density, decreased endothelial cell and cardiomyocyte apoptosis, and in situ differentiation of bone marrow–derived endothelial progenitor cells into endothelial cells. These anatomic findings were accompanied by significant improvements in cardiac function. Conclusions— These findings suggest that downregulation of VEGF may compromise microvascular homeostasis in the myocardium and thereby play a central role in the pathogenesis of DCM.

Hyperhomocyst(e)inemia (HH) is an established independent risk factor for coronary, cerebral and peripheral vascular diseases. Recent studies have indicated that certain cardiovascular risk factors, including diabetes and hypercholesterolemia, impair expression of vascular endothelial growth factor (VEGF) and endogenous angiogenesis. In this study, we investigate the impact of moderate HH on angiogen- esis and VEGF pathway in a mouse model of hindlimb ischemia. Upon induction of unilateral hindlimb ischemia, endogenous angiogenesis, expression of VEGF, and phosphorylation of the VEGF receptor Flk-1 were evaluated in mice heterozygous for a deletion of the cystathionine �-synthase gene (CBS) and compared with those observed in CBS�/� mice. CBS�/� mice exhibit moderate HH, as demonstrated by measuring plasma total homocyst(e)ine (tHcy) levels, which were significantly higher in these animals compared with CBS�/� mice (4.77 � 0.82 vs 2.10 � 0.28, p � 0.01). Twenty-eight days after induction of ischemia, hindlimb blood flow was significantly reduced in CBS�/� mice compared with CBS�/� animals (0.49 � 0.03, n � 12 vs 0.71 � 0.09, n � 10; p � 0.05). In addition, there was a significant negative correlation between plasma homocyst(e)ine levels and the laser Doppler perfusion ratio in CBS�/� mice (p � 0.0087, r �� 0.7171). While VEGF expression and Flk-1 phosphorylation were not impaired in the ischemic muscles of CBS�/� mice, phos- phorylation of the endothelial cell survival factor Akt was significantly inhibited by homocyst(e)ine in a dose-dependent manner in human umbilical vein endothelial cell (HUVECs) in vitro. In conclusion, our findings demonstrate that endogenous angiogenesis is inversely related to plasma levels of homocyst(e)ine in genetically engineered, heterozygous mice with moderate HH. This impairment, however, is not dependent on reduced expression of VEGF or impaired phosphorylation of its recep- tor Flk-1. In contrast, our data suggest that impaired Akt phosphorylation mediates the impairment of angiogenesis associated with HH.

Background— Drug-eluting stents represent a useful strategy for the prevention of restenosis using various antiproliferative drugs. These strategies share the liability of impairing endothelial recovery, thereby altering the natural biology of the vessel wall and increasing the associated risk of stent thrombosis. Accordingly, we tested the hypothesis that local delivery via gene-eluting stent of naked plasmid DNA encoding for human vascular endothelial growth factor (VEGF)-2 could achieve similar reductions in neointima formation while accelerating, rather than inhibiting, reendothelialization. Methods and Results— phVEGF 2-plasmid (100 or 200 μg per stent)–coated BiodivYsio phosphorylcholine polymer stents versus uncoated stents were deployed in a randomized, blinded fashion in iliac arteries of 40 normocholesterolemic and 16 hypercholesterolemic rabbits. Reendothelialization was nearly complete in the VEGF stent group after 10 days and was significantly greater than in control stents (98.7±1% versus 79.0±6%, P 2 , P P Conclusions— Acceleration of reendothelialization via VEGF-2 gene–eluting stents provides an alternative treatment strategy for the prevention of restenosis. VEGF-2 gene–eluting stents may be considered as a stand-alone or combination therapy.

Purpose:To evaluate the feasibility, efficacy, and outcome of endovascular therapy combined with immunosuppression for the treatment of arterial pseudoaneurysms due to Behcet's disease.Methods:Elev...

Background— We investigated whether catheter-based, intramyocardial transplantation of autologous endothelial progenitor cells can enhance neovascularization in myocardial ischemia. Methods and Results— Myocardial ischemia was induced by placement of an ameroid constrictor around swine left circumflex artery. Four weeks after constrictor placement, CD31+ mononuclear cells (MNCs) were freshly isolated from the peripheral blood of each animal. After overnight incubation of CD31+ MNCs in noncoated plates, nonadhesive cells (NA/CD31+ MNCs) were harvested as the endothelial progenitor cell–enriched fraction. Nonadhesive CD31− cells (NA/CD31− MNCs) were also prepared. Autologous transplantation of 10 7 NA/CD31+ MNCs, 10 7 NA/CD31− MNCs, or PBS was performed with a NOGA mapping injection catheter to target ischemic myocardium. In a parallel study, 10 5 human CD34+ MNCs, 10 5 human CD34− MNCs, or PBS was transplanted into ischemic myocardium of nude rats 10 minutes after ligation of the left anterior descending coronary artery. In the swine study, ischemic area by NOGA mapping, Rentrop grade angiographic collateral development, and echocardiographic left ventricular ejection fraction improved significantly 4 weeks after transplantation of NA/CD31+ MNCs but not after injection of NA/CD31− MNCs or PBS. Capillary density in ischemic myocardium 4 weeks after transplantation was significantly greater in the NA/CD31+ MNC group than the control groups. In the rat study, echocardiographic left ventricular systolic function and capillary density were significantly better preserved in the CD34+ MNC group than in the control groups 4 weeks after myocardial ischemia. Conclusions— These favorable outcomes encourage future clinical trials of catheter-based, intramyocardial transplantation of autologous CD34+ MNCs in the setting of chronic myocardial ischemia.

PURPOSE To evaluate the early and midterm outcomes after stent-graft implantation for dissection in the descending thoracic aorta. METHODS Sixteen custom-made endovascular stent-grafts were implanted in 15 patients (10 men; mean age 55.9 +/- 13.7 years, range 32-82) with descending thoracic aortic dissection. Indications for stent-graft implantation were persistent symptoms unresponsive to medical treatment or progressive enlargement of the false lumen. Clinical and imaging surveillance with computed tomography was performed within 1 month of the procedure and at 3 to 6-month intervals in follow-up. RESULTS Endovascular stent-graft implantation at the target site was successful in 14 (93%) patients; 1 device migrated, leaving the false lumen open to flow in the failed case. One (7%) patient who was treated emergently for rupture died suddenly 2 days after the procedure. Over an average follow-up of 31.5 +/- 23.8 months, 1 (7%) patient died and 2 (14%) patients underwent surgical treatment due to recurrent dissection. The remaining 10 patients showed complete thrombosis of the false lumen; in 3, the false lumen completely resolved. CONCLUSIONS Endovascular stent-graft implantation in descending thoracic aortic dissection is a feasible, safe, and effective treatment modality. However, further studies are necessary in a greater number of patients to determine if wider application of this minimally invasive procedure is justified.

A combination of ticlopidine and aspirin has been accepted as the standard antithrombotic regimen after coronary stenting. However, ticlopidine poses serious side effects such as neutropenia or thrombocytopenia. Cilostazol, a cyclic adenosine monophosphate phosphodiesterase inhibitor, is a novel antiplatelet agent with vasodilatory properties. We compared the efficacy and safety of cilostazol plus aspirin (C+A) with ticlopidine plus aspirin (T+A) in elective coronary stenting. Three hundred patients were randomly assigned to receive C+A or T+A 2 days before stenting. The primary end point was a composite of angiographic stent thrombosis, or major cardiac events (death, myocardial infarction, bypass surgery, repeat intervention) at 30 days. The secondary end points were bleeding vascular complications, neutropenia, thrombocytopenia, or side effects requiring discontinuation of the drugs at 30 days. The primary end point was reached in 1.4% in the C+A group and 2.0% in the T+A group (p = 1.0). The rate of bleeding vascular complications was 1.4% in the C+A group and 2.0% in the T+A group (p = 1.0). The rate of drug-related side effects was not statistically different between the 2 groups but slightly higher in the T+A group than in the C+A group (2.7% vs 0.7%, p = 0.37). However, neutropenia was seen in 2 patients only in the T+A group. As a poststenting antithrombotic, C+A is as effective as T+A in preventing major cardiac events including stent thrombosis, and safer in that it does not cause neutropenia despite the fact that there is no statistical difference in the incidence of adverse effects and complications.

A debilitating consequence of diabetes mellitus (DM) is neuropathy which globally affects between 20 -30% of diabetic patients and up to 50% [1, 2]. The lifetime incidence of diabetic neuropathy (DN) is estimated to be up to 45% for type 2 diabetic patients and 59% for type 1 diabetic patients in USA. The risk of DN rises with age, duration of DM, and vascular disease. Characterized by damages in the arms and legs, peripheral neuropathy is the most common complication of DM. The pathophysiology of DN is promoted by several risk factors: microvascular disease, neural hypoxia, and hyperglycemia-induced effects. At the molecular level, the primary cause of diabetic complications is known to be hyperglycemia, which disrupts cellular metabolism by the formation of reactive oxygen species (ROS). In the aspect of nerve functions, ROS formation increases neuron’s susceptibility to damage. In addition, hyperglycemia impedes production of angiogenic and neurotrophic growth factors, which are necessary for normal function of neurons and glial cells and maintenance of vascular structure. The most common presentation of nerve damage due to the effects of hyperglycemia is neuropathic pain. Peripheral neuropathy may cause foot deformities such as hammertoes and unnoticed sores and infections in the numb areas of the foot. Improperly treated infection frequently extends to the bone and requires an amputation of the foot. There have not been any definitive disease-modifying treatments to reverse DN. The current treatment focuses on tight glycemic control which can reduce potential risk factors for further nerve damage and DN-associated pain management. In many studies, deficiency of neurotrophic factors and lack of vascular support have been regarded as key factors in the development DN. Therefore, cell therapy has recently emerged as an attractive therapeutic strategy to meet the needs of both neurotrophic and vascular deficiencies of DN.

Human pluripotent stem cells (hPSCs) hold great promise for treating ischemic heart disease. However, current protocols for differentiating hPSCs either result in low yields or require expensive cytokines.Here we developed a novel two dimensional (2D) stepwise differentiation system that generates a high yield of cardiomyocytes (CMs) from hPSCs without using special cytokines. Initially, undifferentiated hPSCs were transferred onto Matrigel-coated plates without forming embryoid bodies (EBs) for a few days and were cultured in bFGF-depleted human embryonic stem cells (hESCs) medium. When linear cell aggregation appeared in the margins of the hPSC colonies, the medium was changed to DMEM supplemented with 10% fetal bovine serum (FBS). Thereafter when cell clusters became visible, the medium was changed to DMEM with 20% FBS.At about two weeks of culture, contracting clusters began to appear and the number of contracting clusters continuously increased, reaching approximately 70% of all clusters. These clusters were dissociated by two-step enzyme treatment to monolayered CMs, of which ~90% showed CM phenotypes confirmed by an α-myosin heavy chain reporter system. Electrophysiologic studies demonstrated that the hPSC-derived CMs showed three major CM action potential types with 61 to 78% having a ventricular-CM phenotype. This differentiation system showed a clear spatiotemporal role of the surrounding endodermal cells for differentiation of mesodermal cell clusters into CMs. In conclusion, this system provides a novel platform to generate CMs from hPSCs at high yield without using cytokines and to study the development of hPSCs into CMs.

Diabetic neuropathy (DN), the most common complication of diabetes, frequently leads to foot ulcers and may progress to limb amputations. Despite continuous increase in incidence, there is no clinical therapy to effectively treat DN. Pathogenetically, DN is characterized by reduced vascularity in peripheral nerves and deficiency in angiogenic and neurotrophic factors. We will briefly review the pathogenetic mechanism of DN and address the effects and the mechanisms of cell therapies for DN. To reverse the changes of DN, studies have attempted to deliver neurotrophic or angiogenic factors for treatment in the form of protein or gene therapy; however, the effects turned out to be very modest if not ineffective. Recent studies have demonstrated that bone marrow (BM)-derived cells such as mononuclear cells or endothelial progenitor cells (EPCs) can effectively treat various cardiovascular diseases through their paracrine effects. As BM-derived cells include multiple angiogenic and neurotrophic cytokines, these cells were used for treating experimental DN and found to reverse manifestations of DN. Particularly, EPCs were shown to exert favorable therapeutic effects through enhanced neural neovascularization and neuro-protective effects. These findings clearly indicate that DN is a complex disorder with pathogenetic involvement of both vascular and neural components. Studies have shown that cell therapies targeting both vascular and neural elements are shown to be advantageous in treating DN.

Aging is a risk factor for coronary and peripheral artery disease. The role of TNF-α receptors (TNFR1/p55 and TNFR2/p75) in post-acute myocardial infarction (post-AMI) recovery is not well understood. We hypothesized that TNF signaling through its receptors p55 and p75 may be essential in post-AMI regeneration in adult heart. To test our hypothesis we evaluated post-AMI survival, cardiac function, apoptosis, and angiogenesis in AMI model (LAD ligation) in age-matched young (4–8 weeks old) and old (9–14 months old) WT, p75KO, and p55KO mice. In old WTs there was significant fourfold age-associated increase in post-AMI mortality. In young p75KOs post-AMI mortality was approaching mortality in old WTs (35% vs. 45%) whereas >1/2 of old p75KOs (60%) died within 7 days post-AMI. No post-AMI mortality was observed in young p55KOs, and in old p55KOs post-AMI mortality was lower than in young WTs (10% vs. 15%). Compared to young WT, between days 7 and 28 post-AMI functional recovery (% left ventricular (LV) fractional shortening (FS)) was 10% lower in old WTs and young p75KOs (p

The regenerative potential of bone marrow-derived endothelial progenitor cells (EPCs) has been adapted for the treatment of myocardial and limb ischemia via ex vivo expansion. We sought to enhance EPC function by the efficient genetic modification of EPCs in a rat model of myocardial infarction. Peripheral blood EPCs were expanded and transduced, using adeno-associated virus (AAV). AAV-mediated EPC transduction efficacy was 23 ± 1.2%, which was improved by 4.0- to 7.2-fold after pretreatment with the tyrosine kinase inhibitor genistein. Adult rats (n = 7 in each group) underwent myocardial infarction by left anterior descending coronary artery occlusion, and received autologous EPCs transduced by AAV-IGF-1 or AAV-lacZ into the periinfarct area. Echocardiography demonstrated that cardiac function in the IGF-1-EPC group was significantly improved compared with the lacZ-EPC control group 12 weeks after myocardial infarction. In addition, IGF-1-expressing EPCs led to reduced cardiac apoptosis, increased cardiomyocyte proliferation, and increased numbers of capillaries in the periinfarct area. AAV expression was limited to the targeted heart region only. Pretreatment with genistein markedly improved AAV transduction of EPCs. IGF-1-expressing EPCs exhibit favorable cell-protective effects with tissue-limited expression in rat heart postinfarction.

Background— Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence suggested that diabetic neuropathy is causally related to impaired angiogenesis and deficient growth factors. Accordingly, we investigated whether diabetic neuropathy could be reversed by local transplantation of EPCs. Methods and Results— We found that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin-induced diabetic mice but recovered to normal levels after hind-limb injection of bone marrow–derived EPCs. Injected EPCs were preferentially and durably engrafted in the sciatic nerves. A portion of engrafted EPCs were uniquely localized in close proximity to vasa nervorum, and a smaller portion of these EPCs were colocalized with endothelial cells. Multiple angiogenic and neurotrophic factors were significantly increased in the EPC-injected nerves. These dual angiogenic and neurotrophic effects of EPCs were confirmed by higher proliferation of Schwann cells and endothelial cells cultured in EPC-conditioned media. Conclusions— We demonstrate for the first time that bone marrow-derived EPCs could reverse various manifestations of diabetic neuropathy. These therapeutic effects were mediated by direct augmentation of neovascularization in peripheral nerves through long-term and preferential engraftment of EPCs in nerves and particularly vasa nervorum and their paracrine effects. These findings suggest that EPC transplantation could represent an innovative therapeutic option for treating diabetic neuropathy.

Objective— Recently, we have shown that shear stress regulates the angiogenic potential of endothelial cells in vitro by an Angiopoietin-2 (Ang2)–dependent mechanism; however its pathophysiological significance in vivo was not clear. We hypothesized that Ang2 plays an important role in blood flow recovery after arterial occlusion in vivo by regulating angiogenesis and arteriogenesis. Methods and Results— C57Bl/6J mice underwent femoral artery ligation and were injected with a specific Ang2 inhibitor, L1-10, or vehicle for 10 days. Ang2 mRNA was upregulated at day 2, and Ang2 protein was upregulated at day 2, 5, and 7 in the ligated hindlimb. L1-10 treatment significantly blunted blood flow recovery. L1-10 decreased smooth muscle cell coverage of neovessels without affecting capillary density, suggesting a specific role for Ang2 in arteriogenesis. Mechanistically, L1-10 decreased expression of intercellular and vascular cell adhesion molecules as well as infiltrating monocytes/macrophages in the ischemic tissue. Although L1-10 had no effect on the number of CD11b+ cells (monocytes/macrophages) mobilized in the bone marrow, it maintained elevated numbers of circulating CD11b+ cells in the peripheral blood. Conclusions— These results suggest that Ang2 induced in ischemic tissue plays a critical role in blood flow recovery by stimulating inflammation and arteriogenesis.

Since the first experiments of cell transplantation into the heart were performed in the early 1990s, the identification of adult stem cells has triggered attempts to regenerate damaged heart tissue by cellular transplantation. Until recently, a multitude of adult stem or progenitor cells from various tissues have been proposed to meet this end. Bone marrow in particular has emerged as the most promising source for stem and progenitor cells because, besides being the organ of hematopoietic maintenance, it contains a complex assortment of stem and progenitor cells. A large body of provocative experimental evidence for vascular and myocardial regeneration by these cells has generated further enthusiasm for their use. However, many questions remain unanswered in this new field of research regarding the therapeutic potential and the mechanisms responsible for the observed therapeutic effects. In this review, the authors discuss the therapeutic capacity of currently available representative bone marrow-derived stem and progenitor cells for treating ischemic heart diseases.

Emerging evidence has shown that administration of angiogenic growth factors, either as recombinant protein or by gene transfer, can augment tissue perfusion through neovascularization in animal models of myocardial and hindlimb ischemia. Many cytokines have angiogenic activity; one of those that have been best studied in animal models and clinical trials is vascular endothelial growth factor (VEGF). VEGF has been known to be a key regulator of physiologic and pathologic angiogenesis associated with tumor. Recently the effect of VEGF is not restricted to the direct angiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentation of postnatal vasculogenesis in situ. Clinical trials of therapeutic angiogenesis with VEGF in patients with end-stage coronary artery disease have shown increases in exercise time and reductions in anginal symptoms and have provided objective evidence of improved perfusion and left ventricular function. Larger scale placebo-controlled trials with recombinant protein (rhVEGF165) have been limited to intracoronary and intravenous administration and have shown favorable trends in exercise time and angina frequency. Small-scale, placebo-controlled, randomized clinical trials of gene transfer (phVEGF-2) via thoracotomy or percutaneous intramyocardial delivery demonstrated significant improvement of both subjective symptoms and objective measures of myocardial ischemia. Both therapeutic modalities appear to be safe and well tolerated. Further studies are required to determine the optimal dose, formulation, route of administration, and combinations of growth factors and the utility of adjunctive endothelial progenitor cell or other stem cell supplementation, to provide safe and effective therapeutic myocardial neovascularization.

Objective— The embryonic morphogen sonic hedgehog (SHh) has been shown to induce neovascularization of ischemic tissue but has not been shown to play a role in regulating vascular nerve supply. Accordingly, we investigated the hypothesis that systemic injection of SHh protein could improve nerve blood flow and function in diabetic neuropathy (DN). Methods and Results— Twelve weeks after induction of diabetes with streptozotocin, motor and sensory nerve conduction velocities (MCV and SCV) of the sciatic nerves were significantly reduced in diabetic rats. SHh-treated diabetic rats demonstrated marked improvement of both MCV and SCV ( P P Conclusions— These data indicate that Shh induces arteriogenesis and restores nerve function in DN.

Background— There has been a rapid increase in the number of clinical trials using unselected bone marrow (BM) cells or the mononuclear fraction of BM cells for treating ischemic heart diseases. Thus far, no significant deleterious effects or complications have been reported in any studies using BM-derived cells for treatment of various cardiac diseases. Methods and Results— Seven-week-old female Fisher-344 rats underwent surgery to induce acute myocardial infarction and were randomized into 3 groups of 16 rats, each receiving intramyocardial injection of either 7×10 5 DiI-labeled total BM cells (TBMCs), the same number of DiI-labeled, clonally expanded BM multipotent stem cells, or the same volume of phosphate-buffered saline in the peri-infarct area. Echocardiography 2 weeks after cell transplantation indicated intramyocardial calcification in 4 of 14 surviving rats (28.5%) in the TBMC group. Histological examination with hematoxylin and eosin staining and von Kossa staining confirmed the presence of extensive intramyocardial calcification. Alkaline phosphatase staining revealed strong positivity surrounding the calcified area suggestive of ongoing osteogenic activity. Fluorescent microscopic examination revealed that acellular calcific areas were surrounded by DiI-labeled TBMCs, suggesting the direct involvement of transplanted TBMCs in myocardial calcification. In contrast, in hearts receiving equal volumes of saline or BM multipotent stem cells delivered in the same manner, there was no evidence of calcification. Conclusions— These results demonstrate that direct transplantation of unselected BM cells into the acutely infarcted myocardium may induce significant intramyocardial calcification.

mportance Many patients with peripheral artery disease (PAD) have walking impairment despite therapy. Experimental studies in animals demonstrate improved perfusion in ischemic hind limb after mobilization of bone marrow progenitor cells (PCs), but whether this is effective in patients with PAD is unknown. Objective To investigate whether therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) improves exercise capacity in patients with intermittent claudication. Design, Setting, and Participants In a phase 2 double-blind, placebo-controlled study, 159 patients (median [SD] age, 64 [8] years; 87% male, 37% with diabetes) with intermittent claudication were enrolled at medical centers affiliated with Emory University in Atlanta, Georgia, between January 2010 and July 2012. Interventions Participants were randomized (1:1) to received 4 weeks of subcutaneous injections of GM-CSF (leukine), 500 µg/day 3 times a week, or placebo. Both groups were encouraged to walk to claudication daily. Main Outcomes and Measures The primary outcome was peak treadmill walking time (PWT) at 3 months. Secondary outcomes were PWT at 6 months and changes in circulating PC levels, ankle brachial index (ABI), and walking impairment questionnaire (WIQ) and 36-item Short-Form Health Survey (SF-36) scores. Results Of the 159 patients randomized, 80 were assigned to the GM-CSF group. The mean (SD) PWT at 3 months increased in the GM-CSF group from 296 (151) seconds to 405 (248) seconds (mean change, 109 seconds [95% CI, 67 to 151]) and in the placebo group from 308 (161) seconds to 376 (182) seconds (change of 56 seconds [95% CI, 14 to 98]), but this difference was not significant (mean difference in change in PWT, 53 seconds [95% CI, -6 to 112], P?=?.08). At 3 months, compared with placebo, GM-CSF improved the physical functioning subscore of the SF-36 questionnaire by 11.4 (95% CI, 6.7 to 16.1) vs 4.8 (95% CI, -0.1 to 9.6), with a mean difference in change for GM-CSF vs placebo of 7.5 (95% CI, 1.0 to 14.0; P?=?.03). Similarly, the distance score of the WIQ improved by 12.5 (95% CI, 6.4 to 18.7) vs 4.8 (95% CI, -0.2 to 9.8) with GM-CSF compared with placebo (mean difference in change, 7.9 [95% CI, 0.2 to 15.7], P?=?.047). There were no significant differences in the ABI, WIQ distance and speed scores, claudication onset time, or mental or physical component scores of the SF-36 between the groups. Conclusions and Relevance Therapy with GM-CSF 3 times a week did not improve treadmill walking performance at the 3-month follow-up. The improvements in some secondary outcomes with GM-CSF suggest that it may warrant further study in patients with claudication. Trial Registration clinicaltrials.gov Identifier: NCT01041417 Peripheral artery disease (PAD) affects more than 8 million individuals in the United States.1- 3 Although exercise, smoking cessation, antiplatelet therapy, cilostazol, statins, and revascularization are used to treat PAD, men and women with PAD have significantly greater functional impairment and faster functional decline than those without PAD.1,4- 7 Stem and progenitor cell (PC) therapy that promotes neoangiogenesis is an emerging treatment modality in PAD. Progenitor cells, particularly those of endothelial origin, are involved in vascular repair and regeneration.8 They originate primarily but not exclusively from the bone marrow, differentiate into endothelial and other vascular cells, and contribute to neovascularization during tissue repair by direct and paracrine mechanisms.8,9 Endogenous, pharmacologically stimulated, and exogenous PCs contribute to reendothelialization and neovascularization. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate mobilization of hematopoietic and other PCs from the bone marrow.10,11 In the murine hind limb ischemia model, GM-CSF administered by injection or by plasmid transfer augments circulating levels of PCs, increases capillary density, and promotes arteriogenesis.12 Granulocyte-macrophage colony-stimulating factor also augments neoendothelialization of denuded arteries and promotes proliferation, differentiation, and survival of hematopoietic cells, monocytes, and macrophages.9,10,13,14 Based on the observations of improved neovascularization in experimental models with GM-CSF,9 and some equivocal clinical trials,15- 17 we previously completed a phase 1 dose-escalation trial in patients with claudication, which demonstrated the safety of GM-CSF and its ability to mobilize PCs into the circulation. Additionally, we observed a strong trend toward an improvement in exercise duration after 3 months of treatment.18 In this larger phase 2 study, we tested the hypothesis that GM-CSF administration in patients with symptomatic PAD would improve walking performance. In exploratory analyses, we assessed the safety of GM-CSF for patients with claudication.

The methylenetetrahydrofolate reductase (MTHFR) gene has been associated with increased risk for cardiovascular disease in some, but not all studies. Our data sources included a MEDLINE search of the literature published before December 1998, a bibliography review, and expert consultation. Of 23 studies initially identified, 18 (9855 persons) met the inclusion criteria. Information on sample size, study design, Hardy-Weinberg equilibrium, method of genotype determination, plasma folate and homocysteine were abstracted by two reviewers using a standardized protocol. The overall odds ratio of the MTHFR gene on cardiovascular disease was estimated using the Mantel-Haenzel method. From 12 studies with angiographically-confirmed coronary artery disease (CAD), the overall odds ratio (OR) for CAD among those with heterozygous (V/A) was 1.3 (95% CI, 1.1-1.5), while it was 1.4 (1.2-1.6) for the homozygous mutant (V/V) compared to those with homozygous normal (A/A). However, the overall odds ratio for CAD among those with the V/V genotype versus A/A genotype was not statistically significant (OR: 1.1; 95% CI: 0.9-1.3) after excluding three Japanese studies. The corresponding OR for the three Japanese studies was 2.0 (1.6-2.7). For six studies with myocardial infarction (MI), the overall OR of MI was 1.0 (0.8-1.1) for those with the V/A genotype and 0.9 (0.7-1.1) for those with the V/V genotype, respectively; none of these ORs for MI was statistically significant. The MTHFR gene is associated with increased risk for CAD in Japan, but not in other populations.

Diabetic neuropathy (DN) is the most common and disabling complication of diabetes that may lead to foot ulcers and limb amputations. Despite widespread awareness of DN, the only effective treatments are glucose control and pain management. A growing body of evidence suggests that DN is characterized by reduction of vascularity in peripheral nerves and deficiency in neurotrophic and angiogenic factors. Previous studies have tried to introduce neurotrophic or angiogenic factors in the form of protein or gene for therapy, but the effect was not significant. Recent studies have shown that bone marrow (BM)-derived stem or progenitor cells have favorable effects on the repair of cardiovascular diseases. Since these BM-derived stem or progenitor cells contain various angiogenic and neurotrophic factors, these cells have been attempted for treating experimental DN, and turned out to be effective for reversing various manifestations of experimental DN. These evidences suggest that cell therapy, affecting both vascular and neural components, can represent a novel therapeutic option for treatment of clinical DN.

While bone marrow (BM)-derived cells have been comprehensively studied for their propitious pre-clinical results, clinical trials have shown controversial outcomes. Unlike previously acknowledged, more recent studies have now confirmed that humoral and paracrine effects are the key mechanisms for tissue regeneration and functional recovery, instead of transdifferentiation of BM-derived cells into cardiovascular tissues. The progression of the understanding of BM-derived cells has further led to exploring efficient methods to isolate and obtain, without mobilization, sufficient number of cell populations that would eventually have a higher therapeutic potential. As such, hematopoietic CD31(+) cells, prevalent in both bone marrow and peripheral blood, have been discovered, in recent studies, to have angiogenic and vasculogenic activities and to show strong potential for therapeutic neovascularization in ischemic tissues. This article will discuss recent advancement on BM-derived cell therapy and the implication of newly discovered CD31(+) cells.

[bull]| Background- The regenerative potential of bone marrow-derived endothelial progenitor cells (EPCs) has been adapted for the treatment of myocardial and limb ischemia via ex vivo expansion. Biotechnologically, it is plausible to constitute a potential means of addressing enhancement of EPC functions by efficient genetic modification of EPCs. Recombinant adeno-associated virus (rAAV) is currently being recognized as a gene delivery tool, and we recently demonstrated that rAAV vectors are capable of transducing bone marrow cells. Insulin-like growth factor-1 (IGF-1), which belongs to the insulin family of peptides and acts on a variety of functions such as anti-apoptosis, angiogenesis and proliferation of cells.

The aortic dissection is an acute aortic syndrome, caused by an intimal tear and subsequent splitting of the media by the pulsatile blood flow. Though there would be differences in the origin of aortic dissection and therapeutic modalities, the intermediate and long-term prognoses are poor. Endovascular Stent-graft implantation is a revolutionary technique in the treatment of aortic dissection. The endovascular stent grafting in aortic dissection is less invasive and feasible method and is an effective tool for closing the entry site and promoting clot formation, reducing the size of the false lumen. Therefore, endovascular Stent-graft implantation makes possible the desirable remodelling of aorta. We report 33 year-old male with aortic dissection in the thoracic aorta, which was treated with endovascular Stent-graft implantation. Over the favorable remodelling, his dissection was healed completely by the endovascular treatment using Stent-graft. (Korean Circulation J 2000;30(12):1583-1588)

Carotid artery stenting has been accepted as a potential alternative to carotid endarterectomy in patients with significant carotid artery stenosis. The objective of this study was to evaluate the feasibility, safety and long-term outcome of percutaneous stenting of carotid artery stenosis in patients with coexisting symptomatic coronary disease. Between May 1996 and May 1999, we performed carotid artery stenting at 48 lesions in 36 patients with carotid stenosis of 60% and symptomatic coronary artery stenosis. Twenty-one patients (58%) had neurologic symptoms. Carotid stenting was performed in 43 internal, 2 external and 3 common carotid lesions. We used Wallstent in 46 lesions, Palmaz stent in 2 lesions and Microstent II in 1 lesion. Staged or combined coronary intervention was performed in 18 patients (50%) and staged coronary artery bypass surgery was performed in 6 patients (17%). In the other 12 patients (33%), medical treatments were performed. Carotid stenting was successful in all lesions. Simultaneous bilateral carotid stenting was performed in 11 patients (31%). One major and 1 minor stroke developed during the procedure. There were no deaths during the procedures and within 30 days post-procedure. During the follow-up of 14 +/- 7 (3 to 40) months, there were no deaths or neurological events. On follow-up (6 +/- 1 months) angiography and/or duplex sonography of 44 eligible lesions in 32 patients, there were 2 cases of asymptomatic restenosis (4.5%) which developed in Palmaz stents implanted at the external carotid artery and the common carotid artery, respectively. In conclusion, carotid artery stenting in patients with coexistent carotid and coronary artery disease is feasible, safe and shows favorable follow-up outcomes.

Purpose:The purpose of this study was to evaluate the safety, feasibility and effectiveness of an endoluminallyplaced bifurcated stent-graft (Vanguard) for the treatment of infrarenal abdominal aortic aneurysm (AAA). Methods:Transluminal endovascular stent-graft placements were attempted in 29 patients (28 male, mean age 69±7 years) with AAAs involving the common iliac arteries from Aug. 1997 to Jan. 1999. Endovascular therapy was performed in the cardiac catheterization laboratory with epidural anesthesia. One side of the femoral artery was opened by surgical cutdown for the bifurcated stent-graft entry and the other side was punctured percutaneously for the straight stent-graft. Computed tomography and/or intraarterial angiography were performed during an average follow-up of 10 months (2-18 months). Results:Primary success rate was 75.9% (22 of 29 patients) and the overall success rate was 79.3% with successful correction of one perigraft leak. Twenty patients (69.0%) had significant coronary artery disease. There were two technical failure cases, the one was tortuous iliac vessel with spasm, the other was disconnection of the stent-graft connecting portion. Complications related to procedure occurred in 13.8% of patients (4 of 29 patients) and two of these four patients had procedure-related mortality because of acute renal failure following contrast overdose and sepsis after operation. Conclusions:Endovascular treatment of infrarenal AAA with bifurcated stent-graft (Vanguard) is effective, feasible and relatively safe. However, further investigation for the outcome, complication and long-term follow-up are needed. (Korean Circulation J 1999;29(2):907-912)

Background:Previous reports have suggested that alleles at the plasminogen activator inhibitor-1 (PAI-1) gene are associated with increased risk of developing coronary artery disease, including myocardial infarction and stroke through their effect on PAI-1 levels. Method:We attempted to search English literatures for all reports of possible effects of PAI-1 gene on cardiovascular disease in human published prior to November 1998. We used a Mantel-Haenszel method (fixed effect model) and random effect model, respectively, to perform a meta-analysis of 7 case-control studies that provided information related to the effects of PAI-1 gene on risk of cardiovascular disease. Results:From 7 studies for diagnosed cardiovascular disease, the relative frequencies of the three genotypes among controls was (5G/5G) (homozygous normal), 24.5%;(4G/5G) (heterozygous), 48.2%, and (4G/4G) (homozygous for the mutant, 675 GGGG), 27.3%. These relative frequencies in cases were 21.7% for 5G/5G, 48.0% for 4G/5G, and 30.3% for 4G/4G. In fixed effect model, compared with those with genotype (5G/5G), the overall odds ratio (OR) for cardiovascular disease among those with (4G/5G) was 1.12 (95% CI, 0.93 to 1.34), and it was 1.20 (1.01 to 1.44) for the (4G/4G) genotype. For five studies with myocardial infarction as the outcome, the overall OR of myocardial infarction was 1.20 (0.99 to 1.47) for those with (4G/5G) and 1.24 (1.00, 1.54) for those with (4G/4G) genotypes, respectively. Conclusion:Our findings provide support for the weak association between PAI-1 gene and cardiovascular disease, in particular, myocardial infarction. (Korean Circulation J 1999;29(4):366-373)

Background:The real incidence of atherosclerotic lesions in carotid and peripheral arteries in coronary artery disease patients is not well known in Korea. The aim of this study was to prospectively evaluate the prevalence of atherosclerotic involvement of the coronary, carotid, and peripheral arteries in each arterial disease patients. This study was also designed to evaluate the risk factors, the clinical characteristics of associated carotid artery stenosis in patients with coronary artery disease, and associated peripheral vascular disease in patients with coronary artery disease. Methods:Between June 1996 and March 1998, 475 patients (369 males, 106 females, mean age 60±10 years) were studied. Three hundred and seventy-three patients who presented with ischemic symptoms were enrolled in the coronary artery disease group, 81 patients were enrolled in the peripheral vascular disease group due to presenting claudications, and 21 patients were enrolled in the carotid stenosis group due to presenting cerebrovascular symptoms. Coronary angiography was done by the routine method. Carotid angiography was performed at the aortic arch by the digital subtraction angiography method. Peripheral vascular angiography was taken from the suprarenal abdominal aorta to both femoral arteries. Results:1) Risk factors for coronary stenosis, peripheral vascular disease, and carotid stenosis:The risk factors were not different between coronary stenosis, peripheral vascular disease, and carotid stenosis groups, but smoking was more frequent among patients with peripheral vascular disease than in patients with coronary stenosis (p-value=0.001). 2) Coronary artery stenosis and carotid artery stenosis:The mean age of coronary artery patients with carotid stenosis was significantly older (p-value=0.006) than for patients without carotid stenosis. The prevalence of peripheral vascular disease was more common in patients with carotid stenosis than in patients without carotid stenosis. 3) Coronary artery stenosis and peripheral vascular disease:Carotid stenosis was more common inpatients with peripheral vascular disease than in patients without peripheral vascular disease in the coronary stenosis group. 4) Prevalence of coronary, carotid, and peripheral artery disease:In patients with coronary stenosis, the prevalence of carotid stenosis was 13.9% and that of peripheral vascular disease was 29.2%. In patients with peripheral artery stenosis, the prevalence of coronary stenosis was 45.7% and that of carotid artery disease was 33.3%. In patients with carotid stenosis, the prevalence of coronary stenosis was 81.0% and that of peripheral 논문접수일:1999년 5월 6일 심사완료일:1999년 9 월 1일 교신저자:신원흠, 120-752 서울 서대문구 신촌동 연세대학교 의과대학 심혈관센터 심장내과학교실 전화:(02) 361-7049·전송:(02) 393-2041 E-mail:whshimyumc.yonsei.ac.kr

Background and Objectives:For patients with bilateral carotid artery stenosis, simultaneous bilateral carotid endarterectomy is rarely performed due to a higher perioperative risk for death and strokes. We assessed the immediate and long-term outcomes of simultaneous bilateral carotid stenting (SBCS for internal carotid stenosis in patients at high surgical risk. Materials and Methods:We analyzed 10 patients who underwent SBCS for de novo stenoses of both internal carotid arteries (ICA. Included were those who had 60% to 99% stenosis of extracranial ICAs irrespective of neurologic symptoms and had more than 2 risk factors of Mayo grade Ⅲ (medical risks or Ⅳ (neurologic risks. Results:The patients had a mean age of 67±7 years. Technical success was achieved in all lesions. The mean percent diameter stenosis was reduced from 79±13% to 8±8%. A total of 21 Wallstents were deployed at 20 lesions. One patient had a minor stroke just after the procedure which was completely resolved with local injection of urokinase. There were no deaths, major strokes or myocardial infarctions during the 30 day follow-up. Six months imaging studies were available on all 9 eligible patients with 18 lesions by duplex sonography and angiography. Late clinical follow-up at a mean of 15.1±8.1 months revealed no occurrence of neurologic event or death. Conclusion:SBCS is feasible, safe and effective to treat bilateral de novo ICA stenoses in patients at high surgical risk. The procedure, however, is investigational and more experience is required to define its role in the treatment of this patient population. (Korean Circulation J 1999;29( ( ( (2 :898-906