Interrupted slow cooling of mouse cardiac endothelial cell monolayers.

One of the major challenges in the preservation of complex tissues is the cryosensitivity of the endothelium, the single layer of cells lining blood vessels, corneas, and other tissues. The increasing importance of endothelial monolayers in tissue-engineered constructs for transplantation and research warrants the need to develop protocols for the successful cryopreservation of cells in monolayers. In this chapter, we describe a recently published cryopreservation protocol that we developed based on examination of various factors that influence the post-thaw recovery of endothelial monolayers. To efficiently investigate cryopreservation protocol parameters, we employed an interrupted slow-cooling procedure (graded freezing) that allows dissecting loss of cell viability into contributions from slow-cooling injury and rapid-cooling injury. Our optimized protocol involves culturing cells on Rinzl plastic coverslips, using a combination of a penetrating cryoprotectant (5% dimethyl sulfoxide) and a non-penetrating cryoprotectant (6% hydroxyethyl starch), addition of 2% chondroitin sulfate, controlled cooling at 0.2°C/min or 1°C/min, and removal of cryoprotectant immediately after thaw. The protocol has been validated for human umbilical vein and porcine corneal endothelial cell monolayers.

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential

Introduction: Stimulating cardiomyocyte (CM) proliferation is a major strategy for achieving therapeutic heart regeneration. However, heart regeneration requires coordinated interactions of multiple cell types. Because a hallmark of advanced heart failure is vascular rarefaction, the requirement of cardiac endothelial cells (CECs) for cardiac growth and regeneration is of particular importance. Hypothesis: We hypothesized that CECs are required for CM proliferation during growth and regeneration. Methods and Results: We performed a large-scale histologic assessment of neonatal mouse hearts and found the rate of CEC proliferation to shadow CM proliferation over the first 10 days of life. Using a nearest neighbor analysis, we found the fraction of proliferating CECs to be significantly enriched around cycling CMs compared to non-cycling CMs, suggesting that CEC and CM expansion are coupled within a myovascular niche. Single cell sequencing of neonatal mouse hearts after cryoinjury revealed that a majority of these proliferating CECs also express Vegfr2 . To functionally link CEC and CM proliferation, we generated Cdh5-CreER T2 ; Vefgr2 flox/flox mice to genetically delete Vegfr2 from CECs. Compared to mice with intact Vegfr2 , loss of Vegfr2 from CECs in neonatal mice leads to loss of CECs and severely dampens CM proliferation by 4 days (7.01±0.88% vs 0.39±0.35%, p = 7.4x10-4, n = 9),. Interestingly, CM proliferation is attenuated when Vegfr2 is deleted from CECs despite an increase of hypoxia indicators in CMs, signifying that hypoxia-induced CM proliferation is dependent on CECs. In contrast to CEC depletion, treatment of cryoinjured neonatal hearts with AAV encoding the master angiogenic factor, Vegfa can enhance heart regeneration with increased CM cycling in the borderzone (12.6±2.2% vs 5.4±0.4%, p = 0.02, n = 8), reduced scarring of the left ventricle (3.4±1.4% vs 7.6±1.2%, p = 03, n = 16), and improved fractional shortening (51.7±2.5% vs 36.7±4.3%, p = 0.007, n = 14). Conclusions: CEC and CM expansion are spatiotemporally coupled in a myovascular niche during cardiac growth. CECs play a critical role to support CM proliferation and are likely to provide instructive cues that may be leveraged for therapeutic heart regeneration.

Vasoactive agonists like adenosine-5'-triphosphate (ATP) increase intracellular Ca2+ ([Ca2+]i) in vascular endothelial cells with an initial peak due to inositol 1,4,5-triphosphate-mediated Ca2+ release from intracellular stores followed by a sustained plateau that is dependent on the presence of extracellular Ca2+, thus leading to an increased synthesis and release of prostacyclin and nitric oxide. We studied the effects of nucleotides on membrane potential and [Ca2+]i in confluent human microvascular cardiac endothelial cells obtained from patients with dilated cardiomyopathy. The whole-cell configuration of the patch-clamp technique and a confocal laser scanning microscope employing fluo-3 as a Ca2+ indicator were used. Both uridine-5'-triphosphate (UTP) and 2-methylthioadenosine-5'-triphosphate (2MeSATP) induced depolarizations in human microvascular cardiac endothelial cells and increased [Ca2+]i with a rank order of potency 2MeSATP>ATP=UTP (EC50 values (in microM) were 0.084 2MeSATP, 0.67 ATP and 1.1 UTP). This suggests that both P2u and P2y purinoceptors are present on human microvascular cardiac endothelial cells. Maximal [Ca2+]i responses of confluent human microvascular cardiac endothelial cell monolayers to UTP were lower when compared to 2MeSATP. Nucleotide-induced increases in [Ca2+]i consisted of a transient peak, which was also observed in the absence of extracellular Ca2+, and a sustained [Ca2+]i plateau. This plateau, which was not observed in all monolayers studied, was not markedly influenced by increasing extracellular [K+]. Previous incubation with thapsigargin abolished ATP-induced increases of [Ca2+]i. It is concluded that human microvascular cardiac endothelial cells express both P2y and P2u purinoceptors. P2 purinoceptor agonists release Ca2+ from intracellular thapsigargin-sensitive stores and stimulate capacitative Ca2+ influx pathways. K+ efflux through Ca2+-dependent K+ (K(Ca)) channels does not play a major role in the regulation of nucleotide-induced Ca2+ influx in human microvascular cardiac endothelial cells, which might be related to an impaired function of the cells.

Neuregulin-1 (NRG-1), a cardioactive growth factor released from endothelial cells, has been shown to be indispensable for the normal function of the adult heart by binding to ErbB4 receptors on cardiomyocytes. In the present study, we have investigated to what extent ErbB2, the favored co-factor of ErbB4 for heterodimerization, participates in the cardiac effects of endothelium-derived NRG-1. In addition, in view of our previously described anti-adrenergic effects of NRG-1, we have studied which neurohormonal stimuli affect endothelial NRG-1 expression and release and how this may fit into a broader frame of cardiovascular physiology. Immunohistochemical staining of rat heart and aorta showed that NRG-1 expression was restricted to the endocardial endothelium and the cardiac microvascular endothelium (CMVE); by contrast, NRG-1 expression was absent in larger coronary arteries and veins and in aortic endothelium. In rat CMVE in culture, NRG-1 mRNA and protein expression was down-regulated by angiotensin II and phenylephrine and up-regulated by endothelin-1 and mechanical strain. CMVE-derived NRG-1 was shown to phosphorylate cardiomyocyte ErbB2, an event prevented by a 24-h preincubation of myocytes with monoclonal ErbB2 antibodies. Pretreating cardiomyocytes with these inhibitory anti-ErbB2 antibodies significantly attenuated CMVE-induced cardiomyocyte hypertrophy and abolished the protective actions of CMVE against cardiomyocyte apoptosis. Accordingly, ErbB2 signaling participated in the paracrine survival and growth controlling effects of NRG-1 on cardiomyocytes in vitro, explaining the cardiotoxicity of ErbB2 antibodies in patients. Cardiac NRG-1 synthesis occurs in endothelial cells adjacent to cardiac myocytes and is sensitive to factors related to the regulation of blood pressure.

HomeCirculation ResearchVol. 116, No. 2In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published16 Jan 2015https://doi.org/10.1161/RES.0000000000000046Circulation Research. 2015;116:215iCM Reprogramming Factor Stoichiometry (p 237)Wang et al shuffle the gene order in reprogramming vectors and improve fibroblast-to-cardiomyocyte conversion.Download figureDownload PowerPointScar tissue that forms after myocardial infarction reduces heart function and can ultimately lead to heart failure and death. Fibroblasts of the scar tissue, however, can be directly reprogramed into cardiomyocytes—by transfecting the cells with the transcription factors Gata4 (G), Mef2 (M), and Tbx5 (T). While the approach holds great promise for regenerating functional heart muscle its clinical utility is limited by its inefficiency. Getting a precise balance of the three factors might be important for reprogramming efficiency. Thus, Wang et al made six polycistronic vectors—each with the factors in a different possible order (GMT, GTM, MGT and so on)—and compared their reprogramming capacities. They also compared these polycistronic vectors with combined yet separate vectors encoding G, M, and T. Transfection experiments with different vectors showed that each of the constructs produced different ratios of factor expression, and importantly different reprogramming efficiencies. The two constructs in which Mef2 was most 5’—and which happened to have the highest Mef2 expression—reprogrammed fibroblasts more efficiently than other vectors. Indeed, the MGT vector improved reprogramming 10-fold over that seen with individual factor vectors. This improved vector could serve as a platform for further mechanistic studies and optimization, say the authors.Cardiac Exosome Modeling and Therapy (p 255)Gray et al discover that exosomes from hypoxia-treated cardiac progenitor cells promote heart regeneration.Download figureDownload PowerPointTo regenerate heart tissue after a myocardial infarction, researchers have investigated cell therapies involving cardiac progenitor cells. These cells can differentiate into heart cells such as cardiac myocytes, endothelial cells and fibroblasts, but it is not clear whether this differentiation is the sole or even main reason for the improvement of cardiac function after transplantation with progenitor cells. Indeed, there is evidence to support the notion that the paracrine effects of factors secreted by these cells contribute to their beneficial effects. In support of this idea, it has been shown that many cell types, including cardiac progenitors, secrete membrane-bound vesicles called exosomes that can exert certain paracrine effects. Gray and colleagues therefore studied the effects of cardiac progenitor-derived exomes on cardiac cells in vitro and in vivo. To mimic infarction they exposed the progenitors to hypoxia before collecting the exosomes. They showed that compared with exosomes derived under normoxic conditions these hypoxic exosomes induced capillary-like tube growth in cardiac endothelial cells and reduced expression of fibrosis-associated genes in fibroblasts. Moreover these exosomes improved the function of infarcted rat hearts. The team went on to identify specific microRNAs contained in the exosomes that were associated with hypoxia, which could have positive effects on cardiac function and therefore warrant further analysis.CD34+ Cells and Mortality (p 289)Low numbers of circulating progenitor cells indicate high risk of death in coronary artery disease patients, report Patel et al.Download figureDownload PowerPointProgenitor cells, originating primarily from the bone marrow, are important for the regeneration and repair of injured blood vessels. Patel and colleagues therefore hypothesized that a lack of progenitor cells in a person’s blood might indicate an impaired ability for vessel regeneration. To test their hypothesis, the team recruited two cohorts of 502 and 403 patients with coronary artery disease. They counted the progenitor cell numbers in the patients’ blood, and followed their progress for 2.7 years and 1.2 years respectively. The team defined progenitor cells as mononuclear cells expressing the marker CD34. They also examined subsets of these cells that expressed CD133 (a marker of more primitive stem cells), VEGFR2, or CXCR4 (both associated with stem cell recruitment and homing). The team found in both cohorts that the number of CD34+− or CD34+/CD133− cells in a person’s blood was inversely correlated with risk of all-cause death, as well as cardiovascular mortality. Indeed patients with the lowest number of these progenitor cells were almost three times more likely to die. These results suggest not only that progenitor cells may be a useful biomarker for coronary artery disease prognosis, but also that these cells may be useful targets for regenerative therapies. Previous Back to top Next FiguresReferencesRelatedDetails January 16, 2015Vol 116, Issue 2 Advertisement Article InformationMetrics © 2015 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000046 Originally publishedJanuary 16, 2015 PDF download Advertisement

Effect of supercooling and cell volume on intracellular ice formation

Glycosaminoglycan Regulation by VEGFA and VEGFC of the Glomerular Microvascular Endothelial Cell Glycocalyx in Vitro

BackgroundPreservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers.ResultsWe cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution.ConclusionWe conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.

Circulation Research “In This Issue” Anthology

Polymorphonuclear leukocytes (PMNs) play a tremendous role during inflammatory processes. PMNs have to pass a monolayer of endothelial cells to migrate into the extravascular space. Hydroxyethyl starch (HES) is frequently used as a volume expander in critically ill patients. The aim of this study was to investigate whether HES influences the chemotaxis of PMNs through endothelial cell monolayers by using a test system that allows the simultaneous treatment of both cell types. Human umbilical endothelial cells were cultured on microporous membrane filters. PMNs were isolated and PMN chemotaxis was studied. The number of untreated PMNs that migrated through untreated endothelial cell monolayers in response to a chemoattractant was used as a control and set as 100 percent. In clinically relevant concentrations, HES was able to significantly decrease PMN chemotaxis through endothelial cell monolayers, showing a dose-dependent effect (0.1 mg/mL: 99.6 +/- 10.9%, p = NS compared to control; 1 mg/mL: 82.4 +/- 8.3%, p<0.05 compared to control; 10 mg/mL: 62.9 +/- 11.7%, p<0.05). In this assay, both cell types (PMNs and endothelial cells in the monolayer) were treated simultaneously, which simulated the clinical situation after an intravenous injection of HES. The treatment of one cell type, PMNs (89.6 +/- 8.8%, p<0.05) or endothelial cells in the monolayer (76.2 +/- 9.4%, p<0.05), suggests that the influence on endothelial cells is greater. HES is able to significantly reduce the chemotaxis of PMNs through endothelial cell monolayers. The possible clinical consequence of a moderate reduction in endothelium-mediated PMN chemotaxis in critically ill patients remains to be evaluated.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer a powerful in vitro tool to investigate disease mechanisms and to perform patient-specific drug screening. To date, electrophysiological analysis of iPSC-CMs has been limited to single-cell recordings or low-resolution microelectrode array mapping of small cardiomyocyte aggregates. New methods of generating and optically mapping impulse propagation of large human iPSC-CM cardiac monolayers are needed. Our first aim was to develop an imaging platform with versatility for multiparameter electrophysiological mapping of cardiac preparations, including human iPSC-CM monolayers. Our second aim was to create large electrically coupled human iPSC-CM monolayers for simultaneous action potential and calcium wave propagation measurements. A fluorescence imaging platform based on electronically controlled light-emitting diode illumination, a multiband emission filter, and single camera sensor was developed and utilized to monitor simultaneously action potential and intracellular calcium wave propagation in cardiac preparations. Multiple, large-diameter (≥1 cm), electrically coupled human cardiac monolayers were then generated that propagated action potentials and calcium waves at velocities similar to those commonly observed in rodent cardiac monolayers. The multiparametric imaging system presented here offers a scalable enabling technology to measure simultaneously action potential and intracellular calcium wave amplitude and dynamics of cardiac monolayers. The advent of large-scale production of human iPSC-CMs makes it possible to now generate sufficient numbers of uniform cardiac monolayers that can be utilized for the study of arrhythmia mechanisms and offers advantages over commonly used rodent models.

2. Roles of intracellular ice formation, vitrification of cell water, and recrystallization of intracellular ice on the survival of mouse embryos and oocytes

Special research presentation: isolation and characterization of cardiac specific endothelium in murine development and human embryonic stem cell differentiation

S81 INTRODUCTION: Leukocytes play a tremendous role during inflammatory processes. Leukocytes have to pass a monolayer of endothelial cells (ECM) to migrate from the vascular space into the extravascular tissue to avoid microorganisms. During migration, leukocytes undergo morphological changes. Hydroxyethyl starch (HES) is frequently used in cryopreservation, leukapheresis and in infusion as volume expander during trauma. METHODS: Whole blood was taken from healthy volunteers. The neutrophils were isolated using a ficoll-percoll method. Human umbilical vein endothelial cells were isolated and cultured on microporous membranes to achieve a endothelial cell monolayer (ECM). ECM and neutrophils were pre-incubated HES (200/0.5) using different concentrations (1%, 5%, 10%). Leukocyte migration was measured using a previously described assay. In separate experiments only one type of cell, neutrophils or ECM were pre-treated with HES. All experiments were performed n=5 and a p-value < 0.05 were considered as significant. RESULTS: The migration rate of untreated PMNL through untreated ECM was used as control and set as 100 %. In clinically relevant concentrations, HES was able to significantly decrease leukocyte migration through endothelial cell monolayers (HES 10%: 62 +/- 12 % SD, p<0.05). The use of different concentrations of HES showed a dose-dependent effect. In this assay, both cell types (PMNL and ECM) were treated simultaneously, simulating the clinical situation after an i.v. injection. The treatment of one cell type, only PMNL (HES 10%: 89 +/- 9 % SD, p<0.05) or only ECM (HES 10%: 76 +/- 2 % SD, p<0.05), suggests that the influence is rather on endothelial cells than on leukocytes. CONCLUSION: We investigated the cellular interaction between both cell systems simultaneously in the presence of HES. HES is able to reduce significantly the migration of leukocytes through endothelial cell monolayers when both cell systems are pre-treated simultaneously.