Metabolic Syndrome and Cardiac Vessel Remodeling Associated with Vessel Rarefaction: A Possible Underlying Mechanism May Result from a Poor Angiogenic Response to Altered VEGF Signaling Pathways.

BACKGROUND: Elevated mortality rates in patients with metabolic syndrome (MetS) are partly due to adverse remodeling of multiple organs, which may lead to cardiovascular disease, nonalcoholic fatty liver disease, kidney failure, or other conditions. MetS symptoms, such as obesity, hypertension, hyperglycemia, dyslipidemia, associated with insulin and leptin resistance, are recognized as major cardiovascular risk factors that adversely affect the heart. SUMMARY: Pathological cardiac remodeling is accompanied by endothelial cell dysfunction which may result in diminished coronary flow, dysregulated oxygen demand/supply balance, as well as vessel rarefaction. The reduced number of vessels and delayed or inhibited formation of collaterals after myocardial infarction in MetS heart may be due to unfavorable changes in endothelial cell metabolism but also to altered expression of vascular endothelial growth factor molecules, their receptors, and changes in signal transduction from the cell membrane, which severely affect angiogenesis. KEY MESSAGES: Given the established role of cardiac vessel endothelial cells in maintaining tissue homeostasis, defining the molecular background underlying vessel dysfunction associated with impaired angiogenesis is of great importance for future therapeutic purposes. Therefore, the aim of this paper was to present current information regarding vascular endothelial growth factor signaling in the myocardium of MetS individuals.

Extracellular matrix molecules associated with lymphatic vessels in health and disease.

Lymphatic vessels (LyVs), responsible for fluid, solute, and immune cell homeostasis in the body, are closely associated with the adjacent extracellular matrix (ECM) molecules whose structural and functional impact on LyVs is currently more appreciated, albeit not entirely elucidated. These molecules, serving as a platform for various connective tissue cell activities and affecting LyV biology should be considered also as an integral part of the lymphatic system. Any alterations and changes in ECM molecules over the course of disease impair the function and structure of the LyV network. Remodeling of LyV cells, which are components of lymphatic vessel walls, also triggers alterations in ECM molecules and interstitial tissue composition. Therefore, in this review we aimed to present the current knowledge on ECM in tissues and particularly on molecules surrounding lymphatics in normal conditions and in disease.

miR-31-5p-Modified RAW 264.7 Macrophages Affect Profibrotic Phenotype of Lymphatic Endothelial Cells In Vitro.

Cardiac lymphatic vessel (LyV) remodeling as a contributor to heart failure has not been extensively evaluated in metabolic syndrome (MetS). Our studies have shown structural changes in cardiac LyV in MetS that contribute to the development of edema and lead to myocardial fibrosis. Tissue macrophages may affect LyV via secretion of various substances, including noncoding RNAs. The aim of the study was to evaluate the influence of macrophages modified by miR-31-5p, a molecule that regulates fibrosis and lymphangiogenesis, on lymphatic endothelial cells (LECs) in vitro. The experiments were carried out on the RAW 264.7 macrophage cell line and primary dermal lymphatic endothelial cells. RAW 264.7 macrophages were transfected with miR-31-5p and supernatant from this culture was used for LEC stimulation. mRNA expression levels for genes associated with lymphangiogenesis and fibrosis were measured with qRT-PCR. Selected results were confirmed with ELISA or Western blotting. miR-31-5p-modified RAW 264.7 macrophages secreted increased amounts of VEGF-C and TGF-ß and a decreased amount of IGF-1. The supernatant from miR-31-5p-modified RAW 264.7 downregulated the mRNA expression for genes regulating endothelial-to-mesenchymal transition (EndoMT) and fibrosis in LECs. Our results suggest that macrophages under the influence of miR-31-5p show the potential to inhibit LEC-dependent fibrosis. However, more studies are needed to confirm this effect in vivo.

Novel insights into embryonic cardiac macrophages.

Macrophages are vital inhabitants of the developing heart. Nonetheless, their key role is not limited to prenatal processes, as embryo-derived macrophages govern the pool of cardiac macrophages also postnatally. Namely, embryonic cardiac macrophages are of yolk sac-, embryonic monocyte-, and heart-tissue origin. They persist, self-renew and/or are gradually replaced by blood monocytes and assume microenvironment-dependent macrophage phenotypes both in the pre- and postnatal heart. Still, it is during embryonic development that cardiac macrophages gain tissue-specific phenotypes and multifunctional diverse properties. Currently, with the emergence of newer research methods, novel facts about embryonic macrophage ontogeny, lifecycle, and repertoire of functions have been revealed. Meeting the high interest in cardiac macrophages, we present this up-to-date overview of embryonic cardiac macrophages, emphasizing the fundamental concepts and discrepancies related to macrophage characteristics, current research gaps, and potential future developments in this field.

Cardiac lymphatics: state of the art.

PURPOSE OF REVIEW: The beneficial role of cardiac lymphatics in health and disease has begun to be recognized, with both preclinical and clinical evidence demonstrating that lymphangiogenesis is activated in cardiovascular diseases. This review aims to summarize our current understanding of the regulation and impact of cardiac lymphatic remodeling during development and in adult life, highlighting emerging concepts regarding distinguishing traits of cardiac lymphatic endothelial cells (LEC). RECENT FINDINGS: Genetic lineage-tracing and clonal analyses have revealed that a proportion of cardiac LECs originate from nonvenous sources. Further, these sources may vary between different regions of the heart, and could translate to differences in LEC sensitivity to molecular regulators. Several therapeutic approaches have been applied to investigate how lymphatics contribute to resolution of myocardial edema and inflammation in cardiovascular diseases. From these studies have emerged novel insights, notably concerning the cross-talk between lymphatics and cardiac interstitial cells, especially immune cells. SUMMARY: Recent years have witnessed a significant expansion in our knowledge of the molecular characteristics and regulation of cardiac lymphatics. The current body of work is in support of critical contributions of cardiac lymphatics to maintain both fluid and immune homeostasis in the heart.

Multiple roles of cardiac macrophages in heart homeostasis and failure.

Macrophages are essential components of the immune system and play a role in the normal functioning of the cardiovascular system. Depending on their origin and phenotype, cardiac macrophages perform various functions. In a steady-state, these cells play a beneficial role in maintaining cardiac homeostasis by defending the body from pathogens and eliminating apoptotic cells, participating in electrical conduction, vessel patrolling, and arterial tone regulation. However, macrophages also take part in adverse cardiac remodeling that could lead to the development and progression of heart failure (HF) in such HF comorbidities as hypertension, obesity, diabetes, and myocardial infarction. Nevertheless, studies on detailed mechanisms of cardiac macrophage function are still in progress, and could enable potential therapeutic applications of these cells. This review aims to present the latest reports on the origin, heterogeneity, and functions of cardiac macrophages in the healthy heart and in cardiovascular diseases leading to HF. The potential therapeutic use of macrophages is also briefly discussed.

A Comprehensive miRNome Analysis of Macrophages Isolated from db/db Mice and Selected miRNAs Involved in Metabolic Syndrome-Associated Cardiac Remodeling.

Cardiac macrophages are known from various activities, therefore we presume that microRNAs (miRNAs) produced or released by macrophages in cardiac tissue have impact on myocardial remodeling in individuals with metabolic syndrome (MetS). We aim to assess the cardiac macrophage miRNA profile by selecting those miRNA molecules that potentially exhibit regulatory functions in MetS-related cardiac remodeling. Cardiac tissue macrophages from control and db/db mice (an animal model of MetS) were counted and sorted with flow cytometry, which yielded two populations: CD45+CD11b+CD64+Ly6Chi and CD45+CD11b+CD64+Ly6Clow. Total RNA was then isolated, and miRNA expression profiles were evaluated with Next Generation Sequencing. We successfully sequenced 1400 miRNAs in both macrophage populations: CD45+CD11b+CD64+Ly6Chi and CD45+CD11b+CD64+Ly6Clow. Among the 1400 miRNAs, about 150 showed different expression levels in control and db/db mice and between these two subpopulations. At least 15 miRNAs are possibly associated with MetS pathology in cardiac tissue due to direct or indirect regulation of the expression of miRNAs for proteins involved in angiogenesis, fibrosis, or inflammation. In this paper, for the first time we describe the miRNA transcription profile in two distinct macrophage populations in MetS-affected cardiac tissue. Although the results are preliminary, the presented data provide a foundation for further studies on intercellular cross-talk/molecular mechanism(s) involved in the regulation of MetS-related cardiac remodeling.

Assessing functional status of cardiac lymphatics: From macroscopic imaging to molecular profiling.

Here we describe various techniques for visualization of the lymphatic vasculature, particularly in the heart. Addressing macro-, microscopic, and molecular levels of lymphatic organization, we give examples of how to explore the roles of specific antigens/markers expressed in lymphatic vessels and their extracellular matrix as structural and functional elements involved in various biological functions of lymphatics. Some obstacles and technical challenges related to lymphatic visualization are also discussed.

Potential functions of embryonic cardiac macrophages in angiogenesis, lymphangiogenesis and extracellular matrix remodeling.

The role of cardiac tissue macrophages (cTMs) during pre- and postnatal developmental stages remains in many aspects unknown. We aimed to characterize cTM populations and their potential functions based on surface markers. Our in situ studies of immunostained cardiac tissue specimens of murine fetuses (from E11to E17) revealed that a significant number of embryonic cTMs (phenotyped by CD45, CD68, CD64, F4/80, CD11b, CD206, Lyve-1) resided mostly in the subepicardial space, not in the entire myocardial wall, as observed in adult individuals. cTMs accompanied newly developed blood and lymphatic vessels adhering to vessel walls by cellular processes. A subpopulation of CD68-positive cells was found to form accumulations in areas of massive apoptosis during the outflow tract remodeling and shortening. Flow cytometry analysis at E14 and E17 stages revealed newly defined three subpopulations:CD64low, CD64highCD206-and CD64highCD206+. The levels of mRNA expression for genes related to regulation of angiogenesis (VEGFa, VEGFb, VEGFc, bFGF), lymphangiogenesis (VEGFc) and extracellular matrix (ECM) remodeling (MMP13, Arg1, Ym1/Chil3, Retlna/FIZZ1) differed among the selected populations and/or embryonic stages. Our results demonstrate a diversity of embryonic cTMs and their tissue-specific locations, suggesting their various potential roles in regulating angiogenesis, lymphangiogenesis and ECM remodeling.

Microvascular and lymphatic dysfunction in HFpEF and its associated comorbidities.

Heart failure with preserved ejection fraction (HFpEF) is a complex heterogeneous disease for which our pathophysiological understanding is still limited and specific prevention and treatment strategies are lacking. HFpEF is characterised by diastolic dysfunction and cardiac remodelling (fibrosis, inflammation, and hypertrophy). Recently, microvascular dysfunction and chronic low-grade inflammation have been proposed to participate in HFpEF development. Furthermore, several recent studies demonstrated the occurrence of generalized lymphatic dysfunction in experimental models of risk factors for HFpEF, including obesity, hypercholesterolaemia, type 2 diabetes mellitus (T2DM), hypertension, and aging. Here, we review the evidence for a combined role of coronary (micro)vascular dysfunction and lymphatic vessel alterations in mediating key pathological steps in HFpEF, including reduced cardiac perfusion, chronic low-grade inflammation, and myocardial oedema, and their impact on cardiac metabolic alterations (oxygen and nutrient supply/demand imbalance), fibrosis, and cardiomyocyte stiffness. We focus primarily on HFpEF caused by metabolic risk factors, such as obesity, T2DM, hypertension, and aging.

Palliative care for people living with cardiac disease.

Many cardiovascular diseases lead to heart failure, which is a progressive syndrome causing significant distress and limiting the quality of life, despite optimal cardiologic treatment. It is estimated that about 26 000 people in Poland suffer from advanced heart failure, and this number is growing. That is why palliative care (PC) dedicated to people living with end­stage cardiac diseases should be urgently implemented in Poland. Well­organized PC may not only relieve symptoms and improve quality of life in people living with cardiac diseases not responding to treatment but also support patients and their families during the dying process. Palliative care in patients with cardiac diseases should be continued during the end-of-life period. It should be implemented regardless of prognosis, and adjusted to patients' needs. Two approaches to PC are presented in this expert opinion. The first one (generic) is provided by all medical professionals incorporating PC principles into the usual patient care. The second approach, namely, specialized PC, is ensured by a multiprofessional team or at least a PC specialist who received appropriate training in PC. The model of needs-based (not prognosis-based) implementation of PC is discussed in this paper. Symptom control, support in decision-making, and sensitive, open communication are considered integral elements of PC interventions. Medical professionals developing PC in Poland should think about groups of patients with special needs like those with valvular heart disease, grown­up congenital heart disease, and pulmonary arterial hypertension, as well as elderly people. This consensus document presents main recommendations for future PC organization in Poland. Among others, we suggest changing the Polish National Health Fund reimbursement rules regarding PC and improving cardiologist education on PC.

Dignity Therapy as an aid to coping for COPD patients at their end-of-life stage.

INTRODUCTION: Observations indicate that struggling with a burden of an incurable disease such as advanced chronic obstructive pulmonary disease (COPD) may result in the weakening of an individual sense of dignity, and be a source of spiritual suffering. Clinicians providing respiratory care to patients should be open to their spiritual needs, in the belief it may improve coping with the end-of-life COPD. The study aimed to assess overall feasibility and potential benefits of Dignity Therapy (DT) in patients with advanced COPD. MATERIAL AND METHODS: Patients with severe COPD, in whom a DT intervention was implemented according to the protocol established by Chochinov et al. were included into the study. An self-designed questionnaire was applied to assess the patients' satisfaction after intervention. Subsequently, the patients' statements were allocated to specific problem categories, corresponding to the spiritual suffering concerns, as structured by Groves and Klauser. RESULTS: DT was completed in 10 patients, with no unexpected side effects. Satisfaction Questionnaire showed a positive effect of DT on the patient' well-being (3.9 on a 5-point Likert scale). The analyses of the patients' original statements enabled an effective identification of the spiritual suffering and spiritual resources and faced by COPD patients. CONCLUSION: DT is an intervention well received by COPD patients, which may help them in recognising and fulfilling their spiritual needs in the last phase of their life. Information acquired on the patients' resources and spiritual challenges may help clinicians improve their care, especially with regard to supporting their patients at the end-of-life stage.

Three-dimensional nanofibrous polystyrene scaffolds modify macrophage phenotypes and activate macrophage angiogenic potential.

3D scaffolds represent an attractive substrate for studying macrophage activation and modification since they mimic extracellular matrix (ECM). However, macrophage response to such materials, particularly with respect to angiogenic potential is still poorly recognized. Therefore, we investigated the effect of 3D nanofibrous polystyrene scaffolds (NPSs) versus tissue culture polystyrene (TCPS) on THP-1-derived macrophages in various environmental conditions, for example, standard (m0), pro-inflammatory (m1), or anti-inflammatory (m2) with respect to pro-angiogenic potential. There were no differences in the expression of TNF-α and IL-10 mRNAs and respective proteins in cells cultured on NPSs compared with flat polystyrene (TCPS), however, NPSs induced an increased VEGF production by macrophages cultured in m0 and m1 media. Cells cultured in m1, and m2 conditions secreted elevated amounts of TNF-α and IL-10, respectively, irrespective of substrate surface geometry. Each macrophage population contains large, medium, and small cells. Moreover, there were significant differences in the proportion of large to small macrophages depending on the medium composition, that is, in m0, m1, and m2 media these proportions were 1:4, 1:3, and 1:10, respectively. The ultrastructure and the immunoexpression of TNF-α and IL-10 were analyzed under a confocal microscope. The results demonstrated differences in cell ultrastructure and suggested that the larger cells were pro-inflammatory macrophages, while the smaller cells were anti-inflammatory macrophages. In conclusion, NPSs activate macrophage pro-angiogenic potential. In addition, an increase in the proportion of pro-inflammatory macrophages relative to anti-inflammatory ones in a given population favors this potential.

Sulodexide inhibits angiogenesis via decreasing Dll4 and Notch1 expression in mouse proepicardial explant cultures.

Sulodexide (SDX) is a mixed drug containing low-molecular-weight heparin sulfate and dermatan sulfate. It exerts mild anticoagulant action but can also affect leukocytes, macrophages, and cell-cell adhesion and may interact with growth factors although its direct influence on endothelial cells is not well described. Clinically, SDX is used for the treatment of cardiovascular diseases, where it exerts anti-inflammatory and endothelial protective effects. The aim of this study was to determine the influence of SDX on tubule formation and angiogenesis-related proteins' mRNA expression in endothelial cell line C166 and mouse proepicardial explants. C166 cells and explants were stimulated with a proangiogenic cocktail containing bFGF/VEGF-A120 /VEGF-A164 enriched with SDX. After stimulation, the number and morphology of tubules stained with anti-CD31 antibody were examined under confocal microscope and expression of mRNA for VEGF-A, VEGF-B, VEGF-C, bFGF, IGF-1, Dll4, and Notch1 was measured with real-time PCR. In C166 cell line, there was no difference in tubule formation and mRNA expression, but in proepicardial explants, we observed reduction in tubule number and in mRNA level for DLL4 and Notch1 after SDX administration. In conclusion, SDX indirectly inhibits angiogenesis in mouse proepicardial explant cultures but has no direct effect on the C166 endothelial cell line.

Proepicardium: Current Understanding of its Structure, Induction, and Fate.

The proepicardium (PE) is a transitory extracardiac embryonic structure which plays a crucial role in cardiac morphogenesis and delivers various cell lineages to the developing heart. The PE arises from the lateral plate mesoderm (LPM) and is present in all vertebrate species. During development, mesothelial cells of the PE reach the naked myocardium either as free-floating aggregates in the form of vesicles or via a tissue bridge; subsequently, they attach to the myocardium and, finally, form the third layer of a mature heart-the epicardium. After undergoing epithelial-to-mesenchymal transition (EMT) some of the epicardial cells migrate into the myocardial wall and differentiate into fibroblasts, smooth muscle cells, and possibly other cell types. Despite many recent findings, the molecular pathways that control not only proepicardial induction and differentiation but also epicardial formation and epicardial cell fate are poorly understood. Knowledge about these events is essential because molecular mechanisms that occur during embryonic development have been shown to be reactivated in pathological conditions, for example, after myocardial infarction, during hypertensive heart disease or other cardiovascular diseases. Therefore, in this review we intended to summarize the current knowledge about PE formation and structure, as well as proepicardial cell fate in animals commonly used as models for studies on heart development. Anat Rec, 302:893-903, 2019. © 2018 Wiley Periodicals, Inc.

Pentoxifylline inhibits angiogenesis via decreasing Dll4 and Notch1 expression in mouse proepicardial explant cultures.

Pentoxifylline (PTX), a non-specific inhibitor of cAMP phosphodiesterases, is commonly used for treatment of peripheral vascular disorders although its direct action on endothelial cells is not well described. The aim of this study was to determine the influence of PTX on tubule formation and mRNA expression for angiogenesis-related proteins in endothelial cell line C166 and mouse proepicardial explants cultured on collagen. C166 cells and explants were stimulated with proangiogenic cocktail containing bFGF/VEGF-A120/VEGF-A164 and with proangiogenic cocktail enriched with PTX. After stimulation the number and morphology of tubules stained with anti-CD31 antibody was examined under a confocal microscope and expression of mRNA for VEGF-A, VEGF-B, VEGF-C, bFGF, IGF-1, Dll4 and Notch1 was measured with RealTime PCR. In C166 cell line there was no significant difference in tubule formation and mRNA expression, but in proepicardial explants we observed a considerable reduction in tubule number and in mRNA levels for Dll4 and Notch1 after PTX administration. In conclusion, PTX indirectly inhibits angiogenesis in mouse proepicardial explant cultures but has no significant effect on C166 endothelial cell line.

Cells with hematopoietic potential reside within mouse proepicardium.

During embryonic development, hematopoietic cells are present in areas of blood-vessel differentiation. These hematopoietic cells emerge from a specific subpopulation of endothelial cells called the hemogenic endothelium. We have previously found that mouse proepicardium contained its own population of endothelial cells forming a network of vascular tubules. We hypothesize that this EC population contains cells of hematopoietic potential. Therefore, we investigated an in vitro hematopoietic potential of proepicardial cell populations. The CD31+/CD45-/CD71- cell population cultured for 10 days in MethocultTM gave numerous colonies of CFU-GEMM, CFU-GM, and CFU-E type. These colonies consisted of various cell types. Flk-1+/CD31-/CD45-/CD71-, and CD45+ and/or CD71+ cell populations produced CFU-GEMM and CFU-GM, or CFU-GM and CFU-E colonies, respectively. Immunohistochemical evaluations of smears prepared from colonies revealed the presence of cells of different hematopoietic lineages. These cells were characterized by labeling with various combinations of antibodies directed against CD31, CD41, CD71, c-kit, Mpl, Fli1, Gata-2, and Zeb1 markers. Furthermore, we found that proepicardium-specific marker WT1 co-localized with Runx1 and Zeb1 and that single endothelial cells bearing CD31 molecule expressed Runx1 in the proepicardial area of embryonic tissue sections. We have shown that cells of endothelial and/or hematopoietic phenotypes isolated from mouse proepicardium possess hematopoietic potential in vitro and in situ. These results are supported by RT-PCR analyses of proepicardial extract, which revealed the expression of mRNA for crucial regulatory factors for hemogenic endothelium specification, i.e., Runx1, Notch1, Gata2, and Sox17. Our data are in line with previous observation on hemangioblast derivation from the quail PE.

Smoking Status and the Five-Factor Model of Personality: Results of a Cross-Sectional Study Conducted in Poland.

Tobacco smoking is the single most important modifiable factor in increased morbidity and premature mortality. Numerous factors-including genetics, personality, and environment-affect the development and persistence of tobacco addiction, and knowledge regarding these factors could improve smoking cessation rates. This study compared personality traits between never, former, and current smokers, using the Five-Factor Model of Personality in a country with a turbulent smoking reduction process.: In this cross-sectional study, 909 Polish adults completed the Revised Neuroticism-Extraversion-Openness Personality Inventory. Our results showed that current smokers' scores for extraversion, one of the five global dimensions of personality, were higher relative to never smokers. Neuroticism, openness to experience, agreeableness, and conscientiousness did not differ significantly according to smoking status. Facet analysis, which described each dimension in detail, showed that current smokers' activity and excitement seeking (facets of extraversion) scores were higher relative to those of never and former smokers. In turn, current smokers' dutifulness and deliberation (facets of conscientiousness) scores were lower than those found in former and never smokers. Never smokers scored the highest in self-consciousness (a facet of neuroticism) and compliance (a component of agreeableness). The study conducted among Polish individuals showed variation in personality traits according to their smoking status; however, this variation differed from that reported in countries in which efforts to reduce smoking had begun earlier relative to Poland. Knowledge regarding personality traits could be useful in designing smoking prevention and cessation programs tailored to individuals' needs.

Vasculogenesis and Its Cellular Therapeutic Applications.

Vasculogenesis was originally defined by Risau in 1997 [Nature 386: 671-674] as the de novo formation of vessels from endothelial progenitor cells (EPCs), so-called angioblasts. Initially, this process was believed to be related only to embryonic life; however, further studies reported vasculogenesis to occur also in adult tissues. This overview presents the current knowledge about the origin, differentiation and significance of EPCs that have been observed in various diseases, tumors, and reparative processes. We also summarize the knowledge of how to activate these cells for therapeutic purposes and the outcomes of the therapies.