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The human heart has the least regenerative capabilities among tissues and organs, and heart disease continues to be a leading cause of mortality in the industrialized world with insufficient therapeutic options and poor prognosis. Therefore, developing new therapeutic strategies for heart regeneration is a major goal in modern cardiac biology and medicine. Recent advances in stem cell biology and biotechnologies such as human pluripotent stem cells (hPSCs) and cardiac tissue engineering hold great promise for opening novel paths to heart regeneration and repair for heart disease, although these areas are still in their infancy. In this review, we summarize and discuss the recent progress in cardiac tissue engineering strategies, highlighting stem cell engineering and cardiomyocyte maturation, development of novel functional biomaterials and biofabrication tools, and their therapeutic applications involving drug discovery, disease modeling, and regenerative medicine for heart disease.

Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders., (© 2022. The Author(s).)

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole-genome sequencing analysis on 146 nonsyndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this data set with data from a European cohort identified both overlapping and potentially novel gene loci and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene coexpression modules along the cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations.

Background: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role., Methods: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls., Results: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues., Conclusions: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Background: The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics., Methods: A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing., Results: Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes., Conclusion: Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies., (© 2021. The Author(s).)

The combination of population and single-cell RNA sequencing analysis using human embryonic stem cell (hESC) differentiation and developmental tissues is a powerful approach to elucidate an organ-specific cellular and molecular atlas in human embryogenesis. This protocol describes (1) cardiac-directed differentiation and isolation of hESC-derived cardiac derivatives with fluorescence-activated cell sorting, (2) isolation of human embryonic heart-derived single cardiac cells, and (3) construction of cDNA libraries with Smart-seq2. These allow for the preparation of human developmental samples for comprehensive transcriptional analysis. For complete details on the use and execution of this protocol, please refer to Sahara et al. (2019)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA-Seq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis., (©AlphaMed Press 2020.)

The mammalian hearts have the least regenerative capabilities among tissues and organs. As such, heart regeneration has been and continues to be the ultimate goal in the treatment against acquired and congenital heart diseases. Uncovering such a long-awaited therapy is still extremely challenging in the current settings. On the other hand, this desperate need for effective heart regeneration has developed various forms of modern biotechnologies in recent years. These involve the transplantation of pluripotent stem cell-derived cardiac progenitors or cardiomyocytes generated in vitro and novel biochemical molecules along with tissue engineering platforms. Such newly generated technologies and approaches have been shown to effectively proliferate cardiomyocytes and promote heart repair in the diseased settings, albeit mainly preclinically. These novel tools and medicines give somehow credence to breaking down the barriers associated with re-building heart muscle. However, in order to maximize efficacy and achieve better clinical outcomes through these cell-based and/or cell-free therapies, it is crucial to understand more deeply the developmental cellular hierarchies/paths and molecular mechanisms in normal or pathological cardiogenesis. Indeed, the morphogenetic process of mammalian cardiac development is highly complex and spatiotemporally regulated by various types of cardiac progenitors and their paracrine mediators. Here we discuss the most recent knowledge and findings in cardiac progenitor cell biology and the major cardiogenic paracrine mediators in the settings of cardiogenesis, congenital heart disease, and heart regeneration., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The advent of pluripotent stem cell biology and facile genetic manipulation via CRISPR technology has ushered in a new era of human disease models for drug discovery and development. While these precision "super models" hold great promise for tailoring personalized therapy, their full potential and in vivo validation have remained elusive., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Objective: To evaluate the effects of shared decision making using a simple decision aid for opioid prescribing after hysterectomy., Methods: We conducted a prospective quality initiative study including all patients undergoing hysterectomy for benign, nonobstetric indications between March 1, 2018, and July 31, 2018, at our academic institution. Using a visual decision aid, patients received uniform education regarding postoperative pain management. They were then educated on the department's guidelines regarding the maximum number of tablets recommended per prescription and the mean number of opioid tablets used by a similar cohort of patients in a previously published study at our institution. Patients were then asked to choose their desired number of tablets to receive on discharge. Structured telephone interviews were conducted 14 days after surgery. The primary outcome was total opioids prescribed before compared with after implementation of the decision aid. Secondary outcomes included opioid consumption, patient satisfaction, and refill requests after intervention implementation., Results: Of 170 eligible patients, 159 (93.5%) used the decision aid (one patient who used the decision aid was subsequently excluded from the analysis owing to significant perioperative complications), including 110 (69.6%) laparoscopic, 40 (25.3%) vaginal, and eight (5.3%) abdominal hysterectomies. Telephone surveys were completed for 89.2% (n=141) of participants. Student's t-test showed that patients who participated in the decision aid (post-decision aid cohort) were discharged with significantly fewer oral morphine equivalents than patients who underwent hysterectomy before implementation of the decision aid (pre-decision aid cohort) (92±35 vs 160±81, P<.01), with no significant change in the number of requested refills (9.5% [n=15] vs 5.7% [n=14], P=.15). In the post-decision aid cohort, 76.6% of patients (n=121) chose fewer tablets than the guideline-allotted maximum. Approximately 76% of patients (n=102) reported having leftover tablets., Conclusion: This quality improvement initiative illustrates that a simple decision aid can result in a significant decrease in opioid prescribing without compromising patient satisfaction or postoperative pain management.

Synthetic chemically modified mRNAs (modRNA) encoding vascular endothelial growth factor (VEGF) represents an alternative to gene therapy for the treatment of ischemic cardiovascular injuries. However, novel delivery approaches of modRNA are needed to improve therapeutic efficacy in the diseased setting. We hypothesized that cell-mediated modRNA delivery may enhance the in vivo expression kinetics of VEGF protein thus promoting more potent angiogenic effects. Here, we employed skin fibroblasts as a "proof of concept" to probe the therapeutic potential of a cell-mediated mRNA delivery system in a murine model of critical limb ischemia (CLI). We show that fibroblasts pre-treated with VEGF modRNA have the potential to fully salvage ischemic limbs. Using detailed molecular analysis we reveal that a fibroblast-VEGF modRNA combinatorial treatment significantly reduced tissue necrosis and dramatically improved vascular densities in CLI-injured limbs when compared to control and vehicle groups. Furthermore, fibroblast-delivered VEGF modRNA treatment increased the presence of Pax7 + satellite cells, indicating a possible correlation between VEGF and satellite cell activity. Our study is the first to demonstrate that a cell-mediated modRNA therapy could be an alternative advanced strategy for cardiovascular diseases., (Copyright © 2019. Published by Elsevier B.V.)

Soluble CD14 (sCD14) is one of the immunomodulatory factors in breast milk (BM). Although it may be involved in the prevention of atopic symptoms and sensitization to both food and inhalant allergens, conflicting evidence exists concerning its protective effects. In this study, we investigated the relationship between sCD14 in colostrum and 1-month BM, and the development of atopic dermatitis (AD) and sensitization to food and aeroallergens at 9 months of age in infants who were exclusively or almost exclusively breastfed up to 4 months of age. BM samples were collected from lactating mothers who participated in a 2 × 2 factorial, randomized, nontreatment controlled trial study set in Tokyo, which looked at the efficacy of emollients and synbiotics in preventing AD and food allergy in children during the first year of life. A total of 258 colostrum samples and 269 1-month BM samples were analyzed. We found that one-month BM sCD14 levels in the AD group were significantly lower than in the non-AD group. Levels of sCD14 in 1-month BM were not related to allergen sensitization in the overall analysis, but egg white sensitization correlated inversely with 1-month BM sCD14 in infants without AD. The results suggest that sCD14 in BM may be involved in atopic manifestations in early infancy.

The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5 + population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1 + TNNT2 + intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Atopic dermatitis (AD) and food allergy (FA) are common childhood diseases, which may either be interrelated or be the result of skin barrier disruption and gut mucosal dysbiosis. Although some evidence suggests the efficacy of emollients and synbiotics, there is no conclusive evidence on the use of these interventions alone or in combination., Objectives: This study is aimed at identifying the efficacy of emollients and synbiotics in preventing AD and FA in children during the first year of life., Methods: The babies of mothers recruited prenatally received either an emollient, synbiotic, both or neither. The intervention was carried out from birth up to 6 months of age. The age of occurrence of AD and FA were reported in multiple questionnaires at 1, 6, and 9 months and at 1 year of age. AD was diagnosed by a pediatrician at 9 months of age., Results: A -total of 459 babies qualified for the outcome assessment at 1 year of age. Neither the emollient nor the synbiotic showed any effect on reducing the development of AD and FA at 1 year of age., Conclusions: This study did not provide any evidence to show that emollients and synbiotics, alone or in combination are sufficient to prevent the occurrence of AD or FA in children up to 1 year of age., (© 2019 S. Karger AG, Basel.)

Despite the continuous discovery of long noncoding RNAs (lncRNAs) with critical developmental roles, our knowledge of lncRNAs that control cardiac lineage commitment is still limited. In this issue, Guo et al. (2018) report a novel lncRNA-mediated multiprotein complex assembly that directly regulates the key transcriptional programs of murine cardiogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Major cardiovascular events including myocardial infarction (MI) continue to dominate morbidity rates in the developed world. Although multiple device therapies and various pharmacological agents have been shown to improve patient care and reduce mortality rates, clinicians and researchers alike still lack a true panacea to regenerate damaged cardiac tissue. Over the previous two to three decades, cardiovascular stem cell therapies have held great promise. Several stem cell-based approaches have now been shown to improve ventricular function and are documented in preclinical animal models as well as phase I and phase II clinical trials. More recently, the cardiac progenitor cell has begun to gain momentum as an ideal candidate for stem cell therapy in heart disease. Here, we will highlight the most recent advances in cardiac stem/progenitor cell biology in regard to both the basics and applied settings.

Aims: It is still controversial whether bone marrow (BM)-derived endothelial progenitor cells (EPCs) can contribute to vascular repair and prevent the progression of vascular diseases. We aimed to characterize BM-derived EPC subpopulations and to evaluate their therapeutic efficacies to repair injured vascular endothelium of systemic and pulmonary arteries., Methods and Results: BM mononuclear cells of Fisher-344 rats were cultured under endothelial cell-conditions. Early EPCs appeared on days 3-6. Late-outgrowth and very late-outgrowth EPCs (LOCs and VLOCs) were defined as cells forming cobblestone colonies on days 9-14 and 17-21, respectively. Among EPC subpopulations, LOCs showed the highest angiogenic capability with enhanced proliferation potential and secretion of proangiogenic proteins. To investigate the therapeutic effects of these EPCs, Fisher-344 rats underwent wire-mediated endovascular injury in femoral artery (FA) and were concurrently injected intraperitoneally with 60mg/kg monocrotaline (MCT). Injured rats were then treated with six injections of one of three EPCs (1×10(6) per time). After 4weeks, transplanted LOCs, but not early EPCs or VLOCs, significantly attenuated neointimal lesion formation in injured FAs. Some of CD31(+) LOCs directly replaced the injured FA endothelium (replacement ratio: 11.7±7.0%). In contrast, any EPC treatment could neither replace MCT-injured endothelium of pulmonary arterioles nor prevent the progression of pulmonary arterial hypertension (PAH). LOCs modified protectively the expression profile of angiogenic and inflammatory genes in injured FAs, but not in MCT-injured lungs., Conclusion: BM-derived LOCs can contribute to vascular repair of injured systemic artery; however, even they cannot rescue injured pulmonary vasculature under MCT-induced PAH., (Copyright © 2015. Published by Elsevier Ltd.)

The latest discoveries and advanced knowledge in the fields of stem cell biology and developmental cardiology hold great promise for cardiac regenerative medicine, enabling researchers to design novel therapeutic tools and approaches to regenerate cardiac muscle for diseased hearts. However, progress in this arena has been hampered by a lack of reproducible and convincing evidence, which at best has yielded modest outcomes and is still far from clinical practice. To address current controversies and move cardiac regenerative therapeutics forward, it is crucial to gain a deeper understanding of the key cellular and molecular programs involved in human cardiogenesis and cardiac regeneration. In this review, we consider the fundamental principles that govern the "programming" and "reprogramming" of a human heart cell and discuss updated therapeutic strategies to regenerate a damaged heart., (© 2015 The Authors.)

Background: Recent studies have suggested that late-outgrowth endothelial progenitor cells (EPCs) derived from human peripheral blood mononuclear cells (hPBMNCs) might have higher angiogenic potential than classically-defined early-outgrowth EPCs (EOCs). However, it still remains unclear which of "so-called" EPC subpopulations defined in a variety of ways has the highest angiogenic potential., Methods and Results: We classified hPBMNC-derived EPC subpopulations by the time of their emergence in culture. EOCs were defined as attached cells on culture days 3-7. Late-outgrowth EPCs, defined as the cell forming colonies with cobblestone appearance since day 10, were further classified as follows: "moderate"-outgrowth EPCs (MOCs) emerging on days 10-16, "late"-outgrowth EPCs (LOCs) on days 17-23, and "very late"-outgrowth EPCs (VOCs) on days 24-30. Flow cytometry analyses showed the clear differences of hematopoietic/endothelial markers between EOC (CD31(+)VE-cadherin(-)CD34(-)CD14(+)CD45(+)) and LOC (CD31(+)VE-cadherin(+)CD34(+)CD14(-)CD45(-)). We found that LOCs had the highest proliferation and tube formation capabilities in vitro along with the highest expression of angiogenic genes including KDR and eNOS. To investigate the in vivo therapeutic efficacies, each EPC subpopulation was intravenously transplanted into immunocompromised mice (total 4 × 10(5) cells) after unilateral hindlimb ischemia surgery. The LOC-treated mice exhibited significantly-enhanced blood flow recovery (flow ratios of ischemic/non-ischemic leg: 0.99±0.02 [LOC group] versus 0.67 ± 0.07 to 0.78 ± 0.09 [other groups]; P < 0.05) and augmented capillary collateral formation in ischemic leg, which were attributable to their direct engraftment into host angiogenic vessels (approximately 10%) and paracrine effects., Conclusion: hPBMNC-derived late-outgrowth EPCs emerging on culture days 17-23 are superior to other EPC subpopulations with regard to therapeutic angiogenic potential., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC(+)CD31(+)CD34(+)CD14(-)KDR(high) endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR(+) mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.

Aims: Angiotensin-converting enzyme 2 (ACE2) is known as a negative regulator of the renin-angiotensin system. We aimed to determine the roles of ACE2 on the development of vascular diseases., Methods and Results: Using two diversely different models of vascular diseases, hyperlipidaemia-induced atherosclerosis in apolipoprotein E knockout (KO) mice and mechanical injury-induced arterial neointimal hyperplasia in C57Bl6 mice, we examined whether ACE2 deficiency could affect formation of the vascular lesions. ACE2 deficiency resulted in significantly larger vascular lesions in both aortic atherosclerotic plaques and arterial neointima formation, compared with ACE2(+) control. These ACE2-deficient vascular lesions exhibited enhanced accumulation of macrophages into the lesions and proliferation of vascular smooth muscle cells (VSMCs), accompanied with increased angiotensin-II (Ang-II) levels and enhanced expression of vascular inflammation-related genes, including vascular cell adhesion molecule (VCAM)-1, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinase (MMP)9 in aorta/artery tissues. Primary bone marrow macrophages and aortic VSMCs isolated from ACE2 KO mice also displayed enhanced pro-inflammatory responsiveness such as up-regulated gene/protein expression of VCAM-1, MCP-1, and MMP9 to stimulation with tumour necrosis factor-α and Ang-II. The similar phenotype was shown in human macrophages and aortic VSMCs that were transfected with ACE2-specific siRNA. In ACE2-deficient VSMCs, inhibition of c-Jun N-terminal kinase (JNK) by pharmacological blockade with SP600125 or genetic knockdown with JNK-specific siRNA significantly attenuated their pro-inflammatory phenotype., Conclusion: ACE2 deficiency promotes the development of vascular diseases associated with Ang-II-mediated vascular inflammation and activation of the JNK signalling, leading to the notion that ACE2 potentially confers protection against vascular diseases.

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

Most of the mammalian heart is formed from mesodermal progenitors in the first and second heart fields (FHF and SHF), whereby the FHF gives rise to the left ventricle and parts of the atria and the SHF to the right ventricle, outflow tract and parts of the atria. Whereas SHF progenitors have been characterized in detail, using specific molecular markers, comprehensive studies on the FHF have been hampered by the lack of exclusive markers. Here, we present Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) as an FHF marker. Lineage-traced Hcn4+/FHF cells delineate FHF-derived structures in the heart and primarily contribute to cardiomyogenic cell lineages, thereby identifying an early cardiomyogenic progenitor pool. As a surface marker, HCN4 also allowed the isolation of cardiomyogenic Hcn4+/FHF progenitors from human embryonic stem cells. We conclude that a primary purpose of the FHF is to generate cardiac muscle and support the contractile activity of the primitive heart tube, whereas SHF-derived progenitors contribute to heart cell lineage diversification.

Objective: ATP-binding cassette transporter subfamily G member 2 (ABCG2), expressed in microvascular endothelial cells in the heart, has been suggested to regulate several tissue defense mechanisms. This study was performed to elucidate its role in pressure overload-induced cardiac hypertrophy., Methods and Results: Pressure overload was induced in 8- to 12-week-old wild-type and Abcg2-/- mice by transverse aortic constriction (TAC). Abcg2-/- mice showed exaggerated cardiac hypertrophy and ventricular remodeling after TAC compared with wild-type mice. In the early phase after TAC, functional impairment in angiogenesis and antioxidant response in myocardium was found in Abcg2-/- mice. In vitro experiments demonstrated that ABCG2 regulates transport of glutathione, an important endogenous antioxidant, from microvascular endothelial cells. Besides, glutathione transported from microvascular endothelial cells in ABCG2-dependent manner ameliorated oxidative stress-induced cardiomyocyte hypertrophy. In vivo, glutathione levels in plasma and the heart were increased in wild-type mice but not in Abcg2-/- mice after TAC. Treatment with the superoxide dismutase mimetic ameliorated cardiac hypertrophy in Abcg2-/- mice after TAC to the same extent as that in wild-type mice, although cardiac dysfunction with impaired angiogenesis was observed in Abcg2-/- mice., Conclusion: ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

Background: An antianginal K(ATP) channel opener nicorandil has various beneficial effects on cardiovascular systems; however, its effects on pulmonary vasculature under pulmonary arterial hypertension (PAH) have not yet been elucidated. Therefore, we attempted to determine whether nicorandil can attenuate monocrotaline (MCT)-induced PAH in rats., Materials and Methods: Sprague-Dawley rats injected intraperitoneally with 60 mg/kg MCT were randomized to receive either vehicle; nicorandil (5.0 mg·kg(-1)·day(-1)) alone; or nicorandil as well as either a K(ATP) channel blocker glibenclamide or a nitric oxide synthase (NOS) inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME), from immediately or 21 days after MCT injection. Four or five weeks later, right ventricular systolic pressure (RVSP) was measured, and lung tissue was harvested. Also, we evaluated the nicorandil-induced anti-apoptotic effects and activation status of several molecules in cell survival signaling pathway in vitro using human umbilical vein endothelial cells (HUVECs)., Results: Four weeks after MCT injection, RVSP was significantly increased in the vehicle-treated group (51.0±4.7 mm Hg), whereas it was attenuated by nicorandil treatment (33.2±3.9 mm Hg; P<0.01). Nicorandil protected pulmonary endothelium from the MCT-induced thromboemboli formation and induction of apoptosis, accompanied with both upregulation of endothelial NOS (eNOS) expression and downregulation of cleaved caspase-3 expression. Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH. These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME. Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration. Nicorandil activated the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in HUVECs, accompanied with the upregulation of both eNOS and Bcl-2 expression., Conclusions: Nicorandil attenuated MCT-induced vascular endothelial damage and PAH through production of eNOS and anti-apoptotic factors, suggesting that nicorandil might have a promising therapeutic potential for PAH.

Objective: To clarify the impact of breast cancer resistance protein 1 (BCRP1)/ATP-binding cassette transporter subfamily G member 2 (ABCG2) expression on cardiac repair after myocardial infarction (MI)., Methods and Results: The ATP-binding cassette transporter BCRP1/ABCG2 is expressed in various organs, including the heart, and may regulate several tissue defense mechanisms. BCRP1/ABCG2 was mainly expressed in endothelial cells of microvessels in the heart. MI was induced in 8- to 12-week-old wild-type (WT) and Bcrp1/Abcg2 knockout (KO) mice by ligating the left anterior descending artery. At 28 days after MI, the survival rate was significantly lower in KO mice than in WT mice because of cardiac rupture. Echocardiographic, hemodynamic, and histological assessments showed that ventricular remodeling was more deteriorated in KO than in WT mice. Capillary, myofibroblast, and macrophage densities in the peri-infarction area at 5 days after MI were significantly reduced in KO compared with WT mice. In vitro experiments demonstrated that inhibition of BCRP1/ABCG2 resulted in accumulation of intracellular protoporphyrin IX and impaired survival of microvascular endothelial cells under oxidative stress. Moreover, BCRP1/ABCG2 inhibition impaired migration and tube formation of endothelial cells., Conclusions: BCRP1/ABCG2 plays a pivotal role in cardiac repair after MI via modulation of microvascular endothelial cell survival and function.

Objective: We examined whether phosphodiesterase-5 (PDE5) inhibition can promote ischemia-induced angiogenesis., Methods and Results: Unilateral hindlimb ischemia was generated by resecting right femoral artery in wild-type C3H/He mice, treated with either vehicle or a PDE5 inhibitor vardenafil (10 mg/kg per day). Four weeks after surgery, vardenafil significantly enhanced blood flow recovery and augmented capillary collateral formation in ischemic muscle (blood flow ratios of ischemic/nonischemic leg: 0.52+/-0.17 [vehicle] versus 0.92+/-0.09 [vardenafil], P<0.01). Vardenafil upregulated protein expression of vascular endothelial growth factor and hypoxia-inducible factor (HIF)-1 alpha in ischemic muscle and enhanced mobilization of Sca-1/Flk-1-positive endothelial progenitor cells (EPCs) in peripheral blood and bone marrow, contributing to neovascularization. Vardenafil also promoted capillary-like tube formation of human umbilical vein endothelial cells and increased the number of human blood mononuclear cell-derived EPCs in vitro. Furthermore, reporter assays showed that vardenafil and cGMP activated the transactivation activity of HIF-1 under hypoxia. These effects of vardenafil were markedly inhibited by genetic ablation of endothelial nitric oxide synthase, a soluble guanylate cyclase inhibitor, and a protein kinase G inhibitor, respectively., Conclusions: Our results suggest that PDE5 inhibition enhances ischemia-induced angiogenesis with mobilization of EPCs through a protein kinase G-dependent HIF-1/vascular endothelial growth factor pathway. PDE5 inhibition may have a therapeutic potential to treat ischemic cardiovascular diseases.

The Bartonella species have been recently recognized as important causative agents of culture-negative bacterial endocarditis. Antineutrophil cytoplasmic antibodies (ANCAs) have been associated with the spectrum of idiopathic small vessel vasculitis. However, a variety of infections can result in a false-positive ANCA test, and especially subacute bacterial endocarditis (SBE) with the presence of ANCAs occasionally mimics the clinical manifestations of an ANCA-associated vasculitis such as skin purpura and glomerulonephritis. In contrast, noninfectious endocardial involvement is known to be part of the spectrum of the manifestations of the ANCA-associated vasculitis. Therefore, it is crucial to distinguish an ANCA-positive SBE from an ANCA-associated vasculitis with endocardial compromise, because the misdiagnosis of an SBE as an ANCA-associated vasculitis can lead to an inappropriate immunosuppressive therapy with catastrophic consequences. The differential diagnosis is sometimes difficult, especially in the case of culture-negative infective endocarditis with a positive ANCA test. We describe here a case of a culture-negative SBE caused by Bartonellaquintana, accompanied with a positive cytoplasmic ANCA test and clinical findings masquerading as ANCA-associated vasculitis. Both a serological test for Bartonella and polymerase chain reaction restriction fragment length polymorphism analysis were helpful for a correct diagnosis and appropriate treatment., (Copyright 2009 S. Karger AG, Basel.)

Introduction: Recent advances in our understanding of the pathophysiological and molecular mechanisms involved in pulmonary arterial hypertension have led to the development of novel and rational pharmacological therapies. In addition to conventional therapy (i.e., supplemental oxygen and calcium channel blockers), prostacyclin or endothelin receptor antagonists have been recommended as a first-line therapy for pulmonary arterial hypertension. However, these treatments have potential limitations with regard to their long-term efficacy and improvement in survival. Furthermore, intravenous prostacyclin (epoprostenol) therapy, which is recommended by most experts for patients with New York Heart Association (NYHA) functional class IV, is complicated, uncomfortable for patients, and expensive because of the cumbersome administration system. Considering these circumstances, it is necessary to develop additional novel therapeutic approaches that target the various components of this multifactorial disease., Case Report: In this short review, we present an overview of the current treatment options for pulmonary arterial hypertension and describe a case report with primary pulmonary hypertension. A male patient with NYHA functional class IV and showing no response to calcium channel blockers and prostacyclin exhibited significantly improved exercise tolerance and hemodynamics and long-term survival for more than 2.5 years after receiving an oral combination therapy of a phosphodiesterase type 5 inhibitor (sildenafil), phosphodiesterase type 3 inhibitor (pimobendan), and nicorandil., Future Perspective: We also discuss the background and plausible potential mechanisms involved in this case, as well as future perspectives in the treatment of pulmonary arterial hypertension.

Dihydropyrimidine dehydrogenase (DPD) is considered to be a key enzyme affecting the prognosis for patients with colorectal cancer. We investigated whether a correlation exists between the expression of DPD and survival in patients with colorectal cancer. The present study was designed to quantify the DPD level using an enzyme-linked immunosorbent assay in tumors and normal tissue specimens obtained from 22 colorectal cancer patients. There were no significant differences in the preoperative features, neither in the intra- and post-operative findings of patients between the high DPD and low DPD groups in tumor tissue. In patients showing an expression of DPD in tumor tissue, the overall survival in the low DPD group tended to be longer than that in the high DPD group (P = 0.076). In contrast, in patients showing an expression of DPD in normal tissue, no significant difference was observed in the overall survival between the high DPD and low DPD groups (P = 0.358). In patients showing an expression of DPD in tumor tissue, the disease-free survival in the low DPD group was longer than that in the high DPD group (P = 0.046), whereas in patients showing an expression of DPD in normal tissue, no significant difference was seen in the disease-free survival between the high DPD and low DPD groups (P = 0.473). There tended to be a correlation between the DPD expression in tumor tissue and that in adjacent normal tissue (R = 0.390, P = 0.073). Based on these findings, we demonstrated the importance of DPD expression in tumor tissue as a prognostic factor in patients with colorectal cancer.

An increasing number of patients with tetralogy of Fallot (TOF) are reaching older age. We encountered a 75-year-old woman with uncorrected TOF and concomitant severe coronary artery disease (CAD) with congestive heart failure. Her CAD risk factor was hyperlipidemia, which had been untreated. Successful percutaneous coronary interventions have improved her clinical condition and provided long-term survival. Although CAD is considered to be a rare complication in adults with TOF, both strict modification of CAD risk factors and early detection of CAD would be also required in this population, given the residual TOF lesions relating to acute exacerbation of clinical presentation.

In contrast to conventional assumption, recent reports propose the possibility that hematopoietic stem cells (HSCs) may have broader potential to differentiate into various cell types. Here, we tested the pluripotency of HSCs by comparing vascular lesions induced by mechanical injury after bone marrow reconstitution with total bone marrow (TBM) cells, c-Kit+ Sca-1+ Lin- (KSL) cells, or a single HSC cell (Tip-SP CD34-KSL cell, CD34- c-Kit+ Sca-1+ Lin- cell with the strongest dye-efflux activity) harboring green fluorescent protein (GFP). The lesions contained a significant number of GFP-positive cells in the TBM and KSL groups, whereas GFP-positive cells were rarely detected in the HSC group. These results suggest that transdifferentiation of a highly purified HSC seems to be a rare event, if it occurs at all, whereas bone marrow cells including the KSL fraction can give rise to vascular cells that substantially contribute to repair or lesion formation after mechanical injury.

Background/aims: In order to predict liver failure which can lead to death after hepatectomy, a sensitive and specific indicator is needed for liver function. Transcystic duct tube (C-tube) drainage after hepatectomy is thought to be useful in decreasing postoperative complications., Methodology: Conventional serum liver function tests, and total bile acid (TBA) and total bilirubin (T.Bil) concentration levels of bile from a C-tube in 11 hepatectomized patients who underwent C-tube drainage were compared on postoperative day 2 (Day 2) and postoperative day 7 (Day 7)., Results: When serum liver function tests were improving between Day 2 and Day 7, the TBA concentration in bile was increasing in contrast to a decreasing T.Bil concentration. On Day 7, TBA concentrations in the bile in patients without liver cirrhosis or with low ICGR15 values were higher than those in patients with liver cirrhosis or with high ICGR15 values, whereas there were no significant differences between T.Bil bile concentrations in the two groups on Day 7, that is, the measurement of TBA bile concentration might be a faster and more accurate parameter for liver function than that of T.Bil bile concentration., Conclusions: TBA bile concentration obtained from C-tubes was a useful liver function indicator after hepatectomy.

Thymidine phosphorylase (TP) is considered to be a key enzyme affecting the prognosis of patients with advanced gastrointestinal cancer. We tried to demonstrate the correlation of TP expression in tumor tissue and adjacent normal tissue, that is, primary normal tissue. The present study was designed to quantify TP level by enzyme-linked immunosorbent assay (ELISA) in tumor tissue and adjacent normal tissue obtained from 42 hepato-gastrointestinal cancer patients including 15 with gastric, 19 with colorectal and 8 with hepatocellular carcinomas. TP levels in tumor tissues were higher than those in adjacent normal tissues (p<0.001). There was a significant correlation between the expression of TP in tumor tissue and adjacent normal tissue (R=0.711, p<0.001; y=23.420+1.534x). On the other hand, there was no significant correlation between the ratio of tumor to adjacent normal tissue levels of TP (TP T/N) and expression of TP in tumor tissue (R=0.250, p=0.110). Thus, TP expression in tumor tissue may be high in proportion to TP expression in primary tissue. Furthermore, in clinical care, not only TP level in tumor tissue but also TP T/N value should be considered when using anticancer agents that become effective after conversion by TP to the active drug 5-FU.

Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are considered to be key enzymes affecting the prognosis for patients with various cancers. We tried to prove the correlation of TP and DPD expression in hepatocellular carcinoma (HCC) and liver metastasis. We quantified TP and DPD levels by an enzyme-linked immunosorbent assay (ELISA) in the tumor (T) and adjacent normal tissue (N) obtained from 8 HCC patients, and 11 liver metastasis patients together with 9 of their primary cancers. TP levels were higher in the primary cancer, liver metastasis, and HCC compared with each adjacent tissue. TP levels were higher in HCC than in liver metastasis, and TP levels in the adjacent tissues of HCC were also higher than those in adjacent tissues of liver metastasis. TP levels were higher in liver metastasis than in primary cancer, and TP levels in adjacent tissues of liver metastasis were also higher than those in adjacent tissues of primary cancer. However, there were no differences in TP T/N ratio between HCC and liver metastasis, and between primary cancer and liver metastasis. DPD levels were lower in the liver metastasis compared with the adjacent liver tissues, and DPD levels in liver metastasis or its adjacent liver tissues were higher than those in primary cancer or its adjacent tissues. There were no differences in DPD T/N ratio between HCC and liver metastasis, and between primary cancer and liver metastasis. Thus, we demonstrated that TP was highly expressed in liver malignancy. We may be able to increase the success of anticancer chemotherapy for liver malignancy while decreasing the side effects by analysis of T/N ratios in TP, DPD, and TP/DPD in addition to TP expression.

We report a case of malignant peritoneal mesothelioma in a 63-year-old man. Right hemicolectomy, partial ileectomy, partial omentectomy, excision of the abdominal wall, and catheterization for intraperitoneal infusion chemotherapy were performed as surgery. Histopathologically, the tumor was composed of papillary and sheet-like proliferation of atypical cells for which an Alcian Blue digestive test with hyaluronidase was positive. By immunohistochemical staining, the tumor cells were stained against HBME-1 and thrombomodulin antibodies. The final diagnosis was a diffuse malignant mesothelioma of the epithelial type. Sequential adjuvant chemotherapies of cisplatin (ip) plus 5-fluorouracil (iv), cisplatin (iv) plus 5-fluorouracil (iv), and mitomycin C (iv) were administered. He is still alive 46 months after surgery. Moreover, the increase in serum hyaluronic acid levels has been related to tumor volume, and has been useful for clinical follow-up. Secondly, we reviewed major chemotherapy previously described for malignant mesothelioma. The total response rate was 469 of 2,493 cases (18.8%). The response rates with single agent chemotherapy, combination chemotherapy, intraperitoneal or intracavitary chemotherapy, continuous hyperthermic peritoneal perfusion chemotherapy, and immunochemotherapy were 150 of 1,146 cases (13.1%), 209 of 1,019 cases (20.5%), 63 of 133 cases (47.4%), 11 of 13 cases (84.6%), and 36 of 182 cases (19.8%), respectively. Direct exposure of antitumor agent to the peritoneal surface is considered to be most effective against malignant peritoneal mesothelioma.

An extrahepatic portosystemic shunt that has neither liver cirrhosis nor portal hypertension is rare. A 60-year-old Japanese woman who had been suffering chronic liver disease and anemia with mild disorientation was admitted to investigate general fatigue with dizziness and disorientation. The laboratory data revealed mild pancytopenia and liver dysfunction including hyperammoniemia, an increased Indocyanine Green 15-min retention rate, and a decreased Fischer's ratio. Color Doppler ultrasonography, computed tomography, and arterial portography revealed an extrahepatic portosystemic shunt that extended tortuously from the superior mesenteric vein into the inferior vena cava, and decreased blood flow in the main portal vein. Judging from intraoperative measurement of portal pressure and intraoperative portography, shunt ligations were performed at both the efferent portion of shunt from the superior mesenteric vein and the afferent portion of the shunt into the inferior vena cava, and resection of the spleen was also performed. On the postoperative laboratory data, pancytopenia disappeared, and liver function improved. Postoperative abdominal imaging showed increased blood flow in the main portal vein and disappearance of the shunt vessel. Moreover, symptoms present before surgery also disappeared. In conclusion, surgical treatment of extrahepatic portosystemic shunts may result in better postoperative quality of life if it is performed in carefully selected patients.

Background: Although some reports have indicated that acute phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA) can predict the prognosis in patients with acute coronary syndrome, the value of these markers in patients with stable coronary artery disease (CAD) still remains obscure. Therefore, our aim was to determine the prognostic value of inflammatory markers in patients with stable coronary artery disease., Methods and Results: We conducted a prospective cohort study in 140 consecutive patients with stable coronary artery disease who had at least one coronary stenosis more than 50% in diameter seen on diagnostic coronary angiography (CAG). We determined serum levels of the SAA/LDL complex as a new marker in addition to CRP and SAA. Serum levels of the SAA/LDL complex were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). End-points were defined as cardiac death, myocardial infarction, cerebral infarction, and coronary revascularization. End-point events occurred in 21 patients (2 death from myocardial infarction, 2 cerebral infarction, and 17 revascularization). Age (year) (OR = 1.14, CI: 1.05-1.25), diabetes mellitus (OR = 3.50, CI: 1.08-11.40), triglyceride (10mg/dl) (OR = 1.12, CI: 1.01-1.23) and SAA/LDL complex (10 microg/ml) (OR = 2.32, CI: 1.05-4.70) were independently related to the events. A reconstitution experiment suggested that the SAA/LDL complex is derived by oxidative interaction between SAA and lipoproteins., Conclusions: The SAA/LDL complex reflects intravascular inflammation directly and can be a new marker more sensitive than CRP or SAA for prediction of prognosis in patients with stable coronary artery disease.

Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are considered to be key enzymes affecting the prognosis for patients with advanced gastrointestinal cancer. Preoperative examination of TP and DPD expression levels and assessment of these enzymes in inoperable cancer patients may contribute to successful treatment. We tried to prove the correlation of TP and DPD expression in preoperative specimens by endoscopy and in surgical specimens. The present study was designed to quantify TP and DPD levels by enzyme-linked immunosorbent assay (ELISA) in tumor tissue obtained from 30 gastrointestinal cancer patients by preoperative endoscopy and surgery, including 15 gastric and 15 colorectal cancers. Successful cases as those in which cancer cells were demonstrated histologically in preoperative specimens by endoscopy were 12 (success rate: 80%) in gastric cancer patients, and 15 (success rate: 100%) in colorectal cancer patients. In successful cases, there were almost significant correlations in all cases, gastric cancer, and colorectal cancer among the expression of TP, DPD, and TP/DPD ratio in each preoperative specimen by endoscopy and surgical specimen, respectively. On the other hand, in the gastric cancer group, 3 unsuccessful cases resulted in a significant departure from ideal equation compared with 12 successful cases. In actual clinical care, physicians should pay attention to and evaluate carefully the data from endoscopical biopsy specimens in which cancer cells may not be demonstrated histologically. Thus, endoscopic analysis of TP and DPD expression in preoperative or inoperable cancer patients may contribute to successful treatment.

Xenobiotic Phase I and Phase II reactions in hepatocytes occur sequentially and cooperatively during the metabolism of various chemical compounds including drugs. In order to investigate the sequential metabolism of 7-ethoxycoumarin (7EC) as model substrate in vitro, xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 (P450 1A1) and UDP-glucuronosyltransferase 1A6 (UGT1A6) were co-expressed in Saccharomyces cerevisiae AH22. Rat P450 1A1 and yeast NADPH-P450 reductase were expressed on a multicopy plasmid (pGYR1) in the yeast. Rat UGT1A6 cDNA with a yeast alcohol dehydrogenase I promoter and terminator was integrated into yeast chromosomal DNA to achieve the stable expression. Co-expression of P450 1A1 and UGT1A6 in yeast microsomes was confirmed by immunoblot analysis. Protease treatment of the microsomes showed the correct topological orientation of UGT to the membranes. The metabolism of 7EC to 7-hydroxycoumarin (7HC) and its glucuronide in yeast microsomes was analyzed by reverse phase HPLC. In a co-expression system containing 7EC, NADPH and UDP-glucuronic acid, glucuronide formation was detected after a lag phase, following the accumulation of 7HC. In the case of P450 1A1 and UGT1A6, efficient coupling of hydroxylation and glucuronidation in 7EC metabolism was not observed in the co-expression system. This P450 and UGT co-expression system in yeast allows the sequential biotransformation of xenobiotics to be simulated in vitro.

Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are considered to be key enzymes affecting the prognosis for patients with gastric and colorectal cancers. We tried to prove the correlation of TP and DPD expressions in gastric and colorectal cancers. The present study was designed to quantify TP and DPD levels by an enzyme-linked immunosorbent assay (ELISA) in tumors and normal tissues obtained from 16 gastric and 20 colorectal cancer patients. TP and TP/DPD ratio in the tumor specimens were almost all higher than those in each normal tissue, especially for tumors in the progressive state. In the early stage of the colorectal cancer group, DPD in the normal tissues were higher than those in the tumor specimens. There were no significant differences between TP levels in the tumor specimens of the two groups, whereas in stages III and IV, those of the gastric cancer group tended to be higher than those of colorectal cancer group. In stages I and II, DPD levels in the tumor specimens tended to be higher in the gastric cancer group than in the colorectal cancer group. DPD T/N was higher in the gastric cancer group than in the colorectal cancer group. There were no significant differences between TP/DPD ratios in the tumor specimens of the two groups, whereas those in normal tissue were higher in the gastric cancer group than in the colorectal cancer group. We may be able to achieve the successful effects or reduction of side effects of anticancer chemotherapy for gastric and colorectal cancer using the results of this study.

Objectives: Women have higher mortality and frequency of complications compared with men after coronary intervention. Possible differences in coronary atherosclerosis between men and women were investigated., Methods: The left anterior descending arteries of 214 patients (164 men, mean age 62.3 +/- 9.10 years; 50 women, mean age 67.8 +/- 7.76 years) were examined. Lesion length, reference diameter, percentage diameter stenosis and minimal lumen diameter were measured by quantitative coronary angiography. Vessel area, lumen area, percentage area stenosis, and remodeling index were measured by intravascular ultrasonography, and presence of calcification in the lesion was classified. These parameters were compared between men and women., Results: There were no significant differences in quantitative coronary angiography, but intravascular ultrasonography showed calcification was more severe in women, vessel area was significantly smaller in women (13.25 +/- 4.21 vs 15.91 +/- 4.35 mm2, p = 0.004), and remodeling index was significantly lower in women (0.95 +/- 0.13 vs 1.04 +/- 0.18, p = 0.015)., Conclusions: Vessel area measured by intravascular ultrasonography was significantly smaller in women, and calcification was more severe in women. Such factors may be involved in the higher mortality in women.