Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin

The early chick heart tube consists of myocardium and endothelium separated by a myocardially derived basement membrane (MBM). As development proceeds, the endothelium undergoes a transition into mesenchyme in a regionally specific manner; only the atrioventricular (AV) and outflow tract, but not the ventricular endothelium, is transformed into mesenchyme, the progenitor of heart septa and valves. Recent experiments have shown that an EDTA extract of MBM can initiate AV endothelium to form mesenchyme in an in vitro collagen gel culture system. Two-dimensional gel electrophoresis of AV region EDTA extracts showed potentially three isoelectric forms of fibronectin (Fn), while extracts from ventricle contained only two forms. The purpose of the present study was to further investigate the significance of these regional differences by testing of specific myocardial regions (AV vs ventricle) for their ability to induce endothelium to form mesenchyme in vitro, and to immunohistochemically determine if a regionally specific distribution of Fn exists in the MBM that can be correlated with previous electrophoretic data. Embryonic heart regions cultured on three-dimensional collagen gels showed that AV endothelium could only form mesenchyme if cocultured with AV myocardium. Coculture with ventricular myocardial explants did not initiate differentiation of AV endothelium. In contrast, ventricular endothelial cells did not form mesenchyme when cocultured with AV or ventricle myocardium. Immunohistochemical localization of Fn revealed three distinct morphological patterns of distribution in the AV-MBM, i.e., an intense lamina densa staining, diffuse staining in fibrils, and as particles. The Fn localized in particles (0.1 to 0.5 micron in diameter) appeared as a gradient of decreasing concentration extending from the myocardium toward the endothelium. In contrast, no particulate Fn staining was observed in the ventricular region. EDTA extraction selectively depleted the particulate form of Fn. Previous work has shown that this extract, which contains several lower Mr proteins in addition to Fn, is biologically active in initiating mesenchyme formation from AV endothelium in vitro. These results show that a regionally specific interaction of the myocardium with the endothelium is required to initiate the formation of prevalvular mesenchyme. This interaction may be mediated by a multicomponent complex involving Fn and other proteins which appear as a regionally distinct particulate only in areas of endothelial differentiation.     

Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice

Transformation of endocardial endothelial cells into invasive mesenchyme is a critical antecedent of cardiac cushion tissue formation. The message for bone morphogenetic protein (BMP)-2 is known to be expressed in myocardial cells in a manner consistent with the segmental pattern of cushion formation [Development 109(1990) 833]. In the present work, we localized BMP-2 protein in atrioventricular (AV) myocardium in mice at embryonic day (ED) 8.5 (12 somite stage) before the onset of AV mesenchymal cell formation at ED 9.5. BMP-2 protein expression was absent from ventricular myocardium throughout the stages examined. After cellularization of the AV cushion at ED 10.5, myocardial BMP-2 protein expression was diminished in AV myocardium, whereas cushion mesenchymal cells started expressing BMP protein. Expression of BMP-2 in cushion mesenchyme persisted during later stages of development, ED 13.5-16, during valuvulogenesis. Intense expression of BMP-2 persisted in the valve tissue in adult mice. Based on the expression pattern, we performed a series of experiments to test the hypothesis that BMP-2 mediates myocardial regulation of cardiac cushion tissue formation in mice. When BMP-2 protein was added to the 16-18 somite stage (ED 9.25) AV endocardial endothelium in culture, cushion mesenchymal cells were formed in the absence of AV myocardium, which invaded into collagen gels and expressed the mesenchymal marker, smooth muscle (SM) alpha-actin; whereas the endothelial marker, PECAM-1, was lost from the invaded cells. In contrast, when noggin, a specific antagonist to BMPs, was applied together with BMP-2 to the culture medium, AV endothelial cells remained as an epithelial monolayer with little expression of SM alpha-actin, and expression of PECAM-1 was retained in the endocardial cells. When noggin was added to AV endothelial cells cocultured with associated myocardium, it blocked endothelial transformation to mesenchyme. AV endothelium treated with BMP-2 expressed elevated levels of TGFbeta-2 in the absence of myocardium, as observed in the endothelium cocultured with myocardium. BMP-2-supported elevation of TGFbeta-2 expression in endocardial cells was abolished by noggin treatment. These data indicated that BMP signaling is required in and BMP-2 is sufficient for myocardial segmental regulation of AV endocardial cushion mesenchymal cell formation in mice.     

Effect of exogenous interferon on the transplantation reactions of mice]

The authors studied the influence of the serum obtained at various periods after the administration of interferon inductors (New castle disease virus, amino ethylisothiouronium, E. coli endotoxin) on the rate of rejection of the skin or cell transplant of mice C3H and CBA, and also CC57Br. The allogenous skin transplant perished more rapidly; there was also an acceleration of elimination of allogenous lymphoid cells, suppression of colony formation by the cells of allogenous bone marrow in the spleen of the irradiated recipient in administration of the serum obtained at the period of maximal content of interferon induced by the Newcastle disease virus and by amino ethylisothiouronium. The cytotoxic activity of lymphocytes of mice CC57Br against the allogenous target cells rose in the presence of these sera. The serum containing interferon induced with E. coli endotoxin failed to influence the rate of the allotransplant rejection and did not increase the cytotoxic activity of lymphocytes.     

Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field

A hallmark of heart-valve development is the swelling and deposition of extracellular matrix in the heart-valve region. Only myocardium overlying this region can signal to underlying endothelium and cause it to lose cell-cell contacts, delaminate, and invade the extracellular space abutting myocardium and endocardium to form endocardial cushions (EC) in a process known as epithelial to mesenchymal transformation (EMT). The heart-valve myocardium expresses bone morphogenetic protein-2 (Bmp2) coincident with development of valve mesenchyme. BMPs belong to the transforming growth factor beta superfamily (TGF-beta) and play a wide variety of roles during development. We show that conditional ablation of Bmp2 in cardiac progenitors results in cell fate changes in which the heart-valve region adopts the identity of differentiated chamber myocardium. Moreover, Bmp2-deficient hearts fail to induce production and deposition of matrix at the heart-valve-forming region, resulting in the inability of the endothelium to swell and impairing the development of ECs. Furthermore, in collagen invasion assays, Bmp2 mutant endothelium is incapable of undergoing EMT, and addition of BMP2 protein to mutant heart explants rescues this phenotype. Our results demonstrate that Bmp2 is both necessary and sufficient to specify a field of cardiac progenitor cells as the heart-valve-inducing region amid developing atria and ventricles.     

Damage and regeneration of the endothelium of the aorta in experimental hypercholesterolemia

In dynamics of the experimental hypercholesterolemia in rabbits, peculiarities of endothelial regeneration have been studied. Comparison of proliferative activity level in endotheliocytes with structural-functional state of the endothelial monolayer at atherogenesis makes it possible to consider, that the lesion of the endothelium cannot be regarded as an initiating factor for formation of atherosclerotic lesions. Formation of the lesions in the internal lining of the arteries is preceded by certain disorders in permeability of the endothelial barrier at increasing concentration of cholesterin in blood plasma, accompanying with a sharp activation of the cell proliferative activity. When lipid plates and atherosclerotic plaques are already formed, the processes of the endothelial damage and regeneration occur in parallel. The regeneration is ensured with an intensive proliferation and growth of endotheliocytes onto deendotheliolized areas of the damaged intima.     

Quantitative evaluation of the development of isoprenaline-induced heart lesions

The development of heart lesions induced by isoprenaline (ISO) was quantitatively evaluated by 203Hg-Mercurascan (MSC) which was taken up and retained in the damaged cells. After the administration of ISO, the MSC uptake increased immediately at a constant rate, which was independent of the dose of ISO. A higher cardiotoxic effect of increased doses of ISO was caused by prolongation of the time during which the development of heart lesions proceeded. Later, when the damaged cells were subjected to cytolysis, MSC uptake decreased. The blockade of the effect of ISO by methypranol immediately stopped any further increase of MSC uptake. The accumulation of other labelled substances (Neohydrin, HgCl2, CaCl2) in the damaged myocardium was compared with the uptake of MSC. MSC and HgCl2 in particular are suitable for the observation of early changes; relatively less CaCl2 accumulated in the damaged hearts, but it can be used for the detection of more advanced lesions.     

Ca2+ in quality control

Calcium (Ca²⁺) has long been known as a ubiquitous intracellular second messenger, exploited by cells to control processes as diverse as development, proliferation, learning, muscle contraction and secretion. The spatial and temporal patterns of these Ca²⁺-associated signals, as well as their amplitude, is precisely controlled to create gradients of the ion, varying considerably depending on cell type and function. Tuning of intracellular Ca²⁺ is achieved in part by the buffering role of mitochondria, whose unperturbed function is essential for maintaining cellular energy balance. Quality of mitochondria is ensured by the process of targeted autophagy or mitophagy, which depends on a molecular cascade driving the catabolic process of autophagy toward damaged or deficient organelles for elimination via the lysosomal pathway. Nonspecific and targeted autophagy are highly regulated processes fundamental to cell growth and tissue homeostasis, allowing resources to be reallocated in nutrient-deprived cells as well as being instrumental in the repair of damaged organelles or the elimination of those in excess. Given the role of Ca²⁺ signaling in many fundamental cellular processes requiring precise regulation, the involvement of Ca²⁺ in autophagy is still somewhat ill-defined, and only in the past few years has evidence emerged linking the two. This mini-review aims to summarize recent work implicating Ca²⁺ as an important regulator of autophagy, outlining a role for Ca²⁺ that may be even more critical in the regulation of targeted mitochondrial autophagy.     

Condition of the endothelium of the aorta during hypodynamia

The state of the aortic internal lining under conditions of hypodynamia has been studied on 47 white male rats morphometrically and stereologically in film preparations of endothelium. The results of measurements have been treated by means of the moving average method. Significance of differences has been estimated using nonparametric criterion of Wilcoxon--Mann--Whitney. For objective periodization of reactions centring of approximated curves with successive calculation of the variation coefficient of the centered ordinates in every time point has been calculated. Hypokinesia produces manifested changes of phasic character in endothelium. Restoration of the disturbed homeostasis in endothelium is ensured on the bases of both intracellular and cellular regeneration. At late stages (beginning from the 20th day) density in arrangement of endotheliocytes in the layer decreases, as well as the degree of tissue regularity. Polymorphism of endothelium is essentially manifested. Parameters demonstrating destruction and death of cells are rather high. Under such conditions, the response reaction of the arterial wall could acquire the character of developing atherosclerotic lesion.     

3D-model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress

The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell-replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three-dimensional (3D) cell aggregates ("cardiospheres") by putative stem cells isolated from adult dog myocardium using poly-L-ornithine. De novo formation of cardiospheres in growth factor-containing medium occurred over a period of 2-3 weeks, but accelerated to 2-3 days when seeded on poly-L-ornithine. Older cardiospheres developed foci of "beating" cells upon co-culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming "vessel-like" networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post-transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart.     

Potentiation of damaging effects of angiotensin II on the endothelium of the rabbit aorta by a beta adrenergic receptor agonist, isoproterenol

Beta-adrenoceptor agonist isoproterenol potentiates the damaging effect of angiotensin II on rabbit aorta endothelium. Compared to the action of angiotensin II alone the amounts of injured cells, damaged intercellular contacts, cell form change, silver uptake and 125I--LDL uptake are increased under simultaneous action of angiotensin II and isoproterenol. Disturbance in barrier function is associated with the damaged cell contracts and cell death. 125I--LDL uptake increase is due to their accumulation in the adventitia. The simultaneous increase in angiotensin II and beta-adrenoceptor activating agents concentrations can damage endothelium and disturb its barrier function.     

Endocardial endothelium in the rat: junctional organization and permeability

Selective permeability of endocardial endothelium has been suggested as a mechanism underlying the modulation of the performance of subjacent myocardium. In this study, we characterized the organization and permeability of junctional complexes in ventricular endocardial endothelium in rat heart. The length of intercellular clefts viewed en face per unit endothelial cell surface area was lower, and intercellular clefts were deeper in endocardial endothelium than in myocardial vascular endothelium, whereas tight junctions had a similar structure in both endothelia. On this basis, endocardia endothelium. might be less permeable than capillary endothelium. However, confocal scanning laser microscopy showed that intravenously injected dextran 10000 coupled to Lucifer Yellow penetrated first the endocardial endothelium and later the myocardial capillary endothelium. Penetration of dextran 10000 in myocardium occurred earlier through subepicardial capillary endothelium than through subendocardial capillary endothelium. Penetration of tracer might thus be influenced by hydrostatic pressure. Dextran of MW 40000 did not diffuse through either endocardial endothelium or capilary endothelium. The ultrastructure of endocardial endothelium may constitute an adaptation to limit diffusion driven by high hydrostatic pressure in the heart. Differences in paracellular diffusion of dextran 10000 between endocardial endothelium and myocardial vessels, may result from differing permeability properties of the endocardium and underlying myocardium.     

The effect of immune cell-derived exosomes in the cardiac tissue repair after myocardial infarction: Molecular mechanisms and pre-clinical evidence

After a myocardial infarction (MI), the inflammatory responses are induced and assist to repair ischaemic injury and restore tissue integrity, but excessive inflammatory processes promote abnormal cardiac remodelling and progress towards heart failure. Thus, a timely resolution of inflammation and a firmly regulated balance between regulatory and inflammatory mechanisms can be helpful. Molecular- and cellular-based approaches modulating immune response post-MI have emerged as a promising therapeutic strategy. Exosomes are essential mediators of cell-to-cell communications, which are effective in modulating immune responses and immune cells following MI, improving the repair process of infarcted myocardium and maintaining ventricular function via the crosstalk among immune cells or between immune cells and myocardial cells. The present review aimed to seek the role of immune cell-secreted exosomes in infarcted myocardium post-MI, together with mechanisms behind their repairing impact on the damaged myocardium. The exosomes we focus on are secreted by classic immune cells including macrophages, dendritic cells, regulatory T cells and CD4⁺ T cells; however, further research is demanded to determine the role of exosomes secreted by other immune cells, such as B cells, neutrophils and mast cells, in infarcted myocardium after MI. This knowledge can assist in the development of future therapeutic strategies, which may benefit MI patients.     

Extracellular High Mobility Group Box 1 Plays a Role in the Effect of Bone Marrow Mononuclear Cell Transplantation for Heart Failure

Transplantation of unfractionated bone marrow mononuclear cells (BMCs) repairs and/or regenerates the damaged myocardium allegedly due to secretion from surviving BMCs (paracrine effect). However, donor cell survival after transplantation is known to be markedly poor. This discrepancy led us to hypothesize that dead donor BMCs might also contribute to the therapeutic benefits from BMC transplantation. High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes nucleosomes, and also acts as a multi-functional cytokine when released from damaged cells. We thus studied the role of extracellular HMGB1 in the effect of BMC transplantation for heart failure. Four weeks after coronary artery ligation in female rats, syngeneic male BMCs (or PBS only as control) were intramyocardially injected with/without anti-HMGB1 antibody or control IgG. One hour after injection, ELISA showed that circulating extracellular HMGB1 levels were elevated after BMC transplantation compared to the PBS injection. Quantitative donor cell survival assessed by PCR for male-specific sry gene at days 3 and 28 was similarly poor. Echocardiography and catheterization showed enhanced cardiac function after BMC transplantation compared to PBS injection at day 28, while this effect was abolished by antibody-neutralization of HMGB1. BMC transplantation reduced post-infarction fibrosis, improved neovascularization, and increased proliferation, while all these effects in repairing the failing myocardium were eliminated by HMGB1-inhibition. Furthermore, BMC transplantation drove the macrophage polarization towards alternatively-activated, anti-inflammatory M2 macrophages in the heart at day 3, while this was abolished by HMGB1-inhibition. Quantitative RT-PCR showed that BMC transplantation upregulated expression of an anti-inflammatory cytokine IL-10 in the heart at day 3 compared to PBS injection. In contrast, neutralizing HMGB1 by antibody-treatment suppressed this anti-inflammatory expression. These data suggest that extracellular HMGB1 contributes to the effect of BMC transplantation to recover the damaged myocardium by favorably modulating innate immunity in heart failure.     

Progenitor cells in vascular repair

PURPOSE OF REVIEW: A common characteristic of all types of vascular disease is endothelial dysfunction/damage followed by an inflammatory response. Although mature endothelial cells can proliferate and replace damaged cells in the vessel wall, recent findings indicate an impact of stem and progenitor cells in repair process. This review aims to briefly summarize the recent findings in stem/progenitor cell research relating to vascular diseases, focusing on the role of stem/progenitor cells in vascular repair. RECENT FINDINGS: It has been demonstrated that endothelial progenitor cells present in the blood have an ability to repair damaged arterial-wall endothelium. These cells may be derived from a variety of sources, including bone marrow, spleen, liver, fat tissues and the adventitia of the arterial wall. In response to cytokine released from damaged vessel wall and adhered platelets, circulating progenitor cells home in on the damaged areas. It was also reported that the adhered progenitor cells can engraft into endothelium and may differentiate into mature endothelial cells. SUMMARY: Vascular progenitor cells derived from different tissues have an ability to repair damaged vessel, in which the local microenvironment of the progenitors plays a crucial role in orchestrating cell homing and differentiation.     

Induction of an epithelial-mesenchymal transition by an in vivo adheron-like complex

The embryonic vertebrate heart consists of two epithelia: the myocardium and endothelium, separated by the myocardial basement membrane (MBM). The myocardium has been shown to induce endothelial transformation into prevalvular mesenchyme in a temporally and site restricted manner. Previously, we hypothesized that the myocardial-endothelial interaction is mediated in vivo by aggregates of 30-nm particles in the MBM which can be removed by EDTA extraction. These MBM extracts contain fibronectin and other lower Mr proteins and can initiate an epithelial-mesenchymal transition in the AV (atrioventricular canal) endothelium of embryonic chick heart in collagen gel culture. These and other data suggested that the 30-nm multicomponent particles are similar, structurally and compositionally, to multimolecular complexes, termed adherons, secreted by L6 muscle cells in culture. The purpose of this study was to (1) test whether the removal of the 30-nm particles from MBM extracts of embryonic chick hearts would remove the in vitro biological activity and (2) determine if the fractionated MBM extracts can cause AV endothelial cells to follow the same differentiation pathway observed in vivo by monitoring immunohistochemically the cell surface expression of N-CAM. Results showed that centrifugation of extract at 100,000g for 1 hr produced a supernatant fraction that was unable to initiate mesenchyme formation from AV endothelium. However, the resuspended pellet fraction did initiate differentiation of endothelium into mesenchyme. Conditioned medium from L6 skeletal muscle cultures could not substitute for the EDTA extract of embryonic heart. Endothelial cells undergoing the transition to form mesenchyme, both in vivo and in vitro, showed a concomitant decrease in N-CAM staining. This suggested that the pellet-induced formation of migrating cells in the collagen gels is not the result a novel in vitro phenomenon.     

Stem cell transplantation: potential impact on heart failure

Cell transplantation is a promising new modality in treating damaged myocardium after myocardial infarction and in preventing postmyocardial infarction LV remodelling. Two strategies are plausible: the first uses adult tissue stem cells to replace the scar tissues and amend the lost myocardium, whilst the second strategy uses embryonic stem cells in an attempt to regenerate myocardium and/or blood vessels.     

Initial expression of type I procollagen in chick cardiac mesenchyme is dependent upon myocardial stimulation

Formation of the atrioventricular (AV) mesenchyme is a critical step in early heart development. Endothelial cells are activated and transformed into a mesenchymal population that invades the cell-free myocardial basement membrane. This process can be duplicated in collagen gel culture, where it has been established that myocardium or its secretory products activate the endothelium. The purpose of the present study was to determine when these activated endothelial and/or mesenchymal cells start producing type I collagen in situ. These results were compared to those obtained from a culture model of mesenchyme formation. The production of type I collagen was monitored using a monoclonal antibody (M38) that recognizes the carboxy-terminal propeptide of human type I procollagen. The initial expression of the latter within activated AV endothelial and mesenchymal cells in ovo was 48 hr following activation. Prior to this time, only the myocardium was reactive with M38. AV explants of early hearts on collagen gels revealed staining of activated endothelial and mesenchymal cells with M38 after 48 hr in coculture with myocardial tissue. Explants that were prevented from activating (myocardium removed) never expressed the M38 antigen. Similarly, AV endothelial monolayers grown in the presence of myocardial conditioned medium activated and expressed type I collagen after 48 hr in culture, whereas those grown in standard medium did not. These results establish the initial expression of type I collagen within activated AV endothelium and mesenchyme. In addition, the data suggest that the expression of type I collagen within the AV mesenchyme may be dependent on extrinsic influences that induce the AV endothelium to transform into mesenchyme.     

Dynamic induction of ADAMTS1 gene in the early phase of acute myocardial infarction

Extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteases (MMPs) play an essential role in the repair of infarcted tissue, which affects ventricular remodeling after myocardial infarction. ADAMTS1 (A disintegrin and metalloprotease with thrombospondin motifs), a newly discovered metalloprotease, was originally cloned from a cancer cell line, but little is known about its contribution to disease. To test the hypothesis that ADAMTS1 appears in infarcted myocardial tissue, we examined ADAMTS1 mRNA expression in a rat myocardial infarction model by Northern blotting, real-time RT-PCR and in situ hybridization. Normal endothelium expressed little ADAMTS1 mRNA, while normal myocardium expressed no detectable ADAMTS1 mRNA. Up-regulation of ADAMTS1 was demonstrated by Northern blot analysis and real-time RT-PCR at 3 h after coronary artery ligation. In situ hybridization revealed strong ADAMTS1 mRNA signals in the endothelium and myocardium in the infarcted heart, mainly in the infarct zone, at 3 h after myocardial infarction. The rapid and transient up-regulation of the ADAMTS1 gene in the ischemic heart was distinct from the regulatory patterns of other MMPs. Our study demonstrated that the ADAMTS1 gene is a new early immediate gene expressed in the ischemic endothelium and myocardium.