assessment-right-ventricular-structure-function-mouse-model-pulmonary

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

ultrasound-based-assessment-coronary-artery-flow-coronary-flow

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

The cell-substrate distance is a direct indicator of cell adhesion to extracellular matrix which is indispensable in cell culture. A real-time monitoring approach can provide a detailed profile of cell adhesion, so that enables the detecting of adhesion-related cell behavior. In this work, we report a novel real-time impedance-based method to record the adhesion profile of cardiomyocyte, overcoming its inscrutability due to the primary culture. Microfabricated biosensors are applied in cardiomyocyte culture after characterizing the cell-free system. Cyclic frequency scanning data of cell-related impedance are generated and automatically fit into the equivalent circuit model, which is established using electrochemical impedance spectroscopy. The data are displayed as the alteration of normalized cell-substrate distance and the essential parameters for manual electric cell-substrate impedance sensing calibration of absolute distance. The time course displays a significant decline in the equivalent cell-substrate distance, from 155.8 to 60.2 nm in the first 20 h of cardiomyocyte culture. Furthermore, the cardiomyocytes cultured in long-term medium and short-term medium (ACCT) for 10 h exhibit distinct difference in adhesion rate as well as cell-substrate distance (72 vs 68 nm).

Real-time monitoring primary cardiomyocyte adhesion based on electrochemical impedance spectroscopy and electrical cell-substrate impedance sensing.

Deregulated cardiomyocyte death is a critical risk factor in a variety of cardiovascular diseases. Although various assays have been developed to detect cell responses during cell death, the capability of monitoring cell detachment will enhance the understanding of death processes by providing instant information at its early phase. In this work, we developed an impedance-sensing assay for real-time monitoring of cardiomyocyte death induced by tumor necrosis factor-alpha based on recording the change in cardiomyocyte adhesion to extracellular matrix. Electrochemical impedance spectroscopy was employed in impedance data processing, followed by calibration with the electrical cell-substrate impedance-sensing technique. The adhesion profile of cardiomyocytes undergoing cell death processes was recorded as the time course of equivalent cell-substrate distance. The cell detachment was detected with our assay and proved related to cell death in the following experiments, indicating its advantage against the conventional assays, such as Trypan blue exclusion. An optimal concentration of tumor necrosis factor-alpha (20 ng/mL) was determined to induce cardiomyocyte apoptosis rather than the combinative cell death of necrosis and apoptosis by comparing the concentration-related adhesion profiles. The cardiomyocytes undergoing apoptosis experienced an increase of cell-substrate distance from 59.1 to 89.2 nm within 24 h. The early change of cell adhesion was proved related to cardiomyocyte apoptosis in the following TUNEL test at t = 24 h, which suggested the possibility of early and noninvasive detection of cardiomyocyte apoptosis.

Impedance-based monitoring of ongoing cardiomyocyte death induced by tumor necrosis factor-alpha.

Cardiomyocyte death caused by proinflammatory cytokines, such as Tumor necrosis factor alpha (TNF-alpha), is one of the hot topics in cardiovascular research. TNF-alpha can induce multiple cell processes that are dependent on the treatment time although the long-term treatment definitely leads to cell death. The ability to intervene in cell death will be invaluable to reveal the effects of short-term TNF-alpha treatment to cardiomyocytes. However, a real-time monitoring technique is needed to guide the intervention of cell responses. In this work, we employed the impedance-sensing technique to real-time monitor the equivalent cell-substrate distance of cardiomyocytes via electrochemical impedance spectroscopy (EIS) and electrical cell-substrate impedance sensing (ECIS). In the stabilized cardiomyocyte culture, the sustained TNF-alpha treatment caused strengthened cell adhesion in the first 2 h which was followed by the transition to cell detachment afterwards. Considering cell detachment was an early morphological evidence of cell death, we removed TNF-alpha from the cardiomyocyte culture before the transition to achieve the intervention of cell responses. The result of this intervention showed that cell adhesion was continuously strengthened before and after the removal of TNF-alpha, indicating the short-term treated cardiomyocytes did not undergo death processes. It was also demonstrated in TUNEL and TBE tests that the percentages of apoptosis and cell death were both lowered.

Intervention of cardiomyocyte death based on real-time monitoring of cell adhesion through impedance sensing.

Cardiomyocyte death is a critical risk factor in a variety of cardiovascular diseases [1, 2]. At the level of individual cardiomyocytes, cell adhesion alters at the early phase of ongoing death. It suggested the possibility of detecting the early sign of cell death through cell detachment and so that we could intervene in the cell response before cell fell into step with the irreversible death processes. In this work, we demonstrated the application of our impedance-sensing technique [3] in the intervention of cell responses to chemical stimuli. The results showed that the cell detachment could be detected with our impedance-sensing technique earlier than conventional biochemical staining techniques, and it could be used to guide the precautionary removal of TNF-alpha from the cell culture to intervene in the cell responses and prevent cardiomyocytes from cell death.

Intervention of cardiomyocyte death based on the impedance-sensing technique of monitoring cell adhesion.

Cell-based microarrays are being increasingly used as a tool for combinatorial and high throughput screening of cellular microenvironments. Analysis of microarrays requires several steps, including microarray imaging, identification of cell spots, quality control, and data exploration. While high content image analysis, cell counting, and cell pattern recognition methods are established, there is a need for new postprocessing and quality control methods for cell-based microarrays used to investigate combinatorial microenvironments. Previously, microarrayed cell spot identification and quality control were performed manually, leading to excessive processing time and potentially resulting in human bias. This work introduces an automated approach to identify cell-based microarray spots and spot quality control. The approach was used to analyze the adhesion of murine cardiac side population cells on combinatorial arrays of extracellular matrix proteins. Microarrays were imaged by automated fluorescence microscopy and cells were identified using open-source image analysis software (CellProfiler). From these images, clusters of cells making up single cell spots were reliably identified by analyzing the distances between cells using a density-based clustering algorithm (OPTICS). Naïve Bayesian classifiers trained on manually scored training sets identified good and poor quality spots using spot size, number of cells per spot, and cell location as quality control criteria. Combined, the approach identified 78% of high quality spots and 87% of poor quality spots. Full factorial analysis of the resulting microarray data revealed that collagen IV exhibited the highest positive effect on cell attachment. This data processing approach allows for fast and unbiased analysis of cell-based microarray data.

Spot identification and quality control in cell-based microarrays.

The heart possesses a regeneration potential derived from endogenous and exogenous stem and progenitor cell populations, though baseline regeneration appears to be sub-therapeutic. This limitation was initially attributed to a lack of cells with cardiomyogenic potential following an insult to the myocardium. Rather, recent studies demonstrate increased numbers of cardiomyocyte progenitor cells in diseased hearts. Given that the limiting factor does not appear to be cell quantity but rather repletion of functional cardiomyocytes, it is crucial to understand potential mechanisms inhibiting progenitor cell differentiation. One of the extensively studied areas in heart disease is extracellular matrix (ECM) remodeling, with both the composition and mechanical properties of the ECM undergoing changes in diseased hearts. This review explores the influence of ECM properties on cardiomyogenesis and adult cardiac progenitor cells.

Regeneration in heart disease-Is ECM the key?

Rationale: Following cardiac injury, cardiac progenitor cells are acutely reduced, and are replenished in part by regulated self-renewal and proliferation, which occurs through symmetric and asymmetric cellular division. Understanding the molecular cues controlling progenitor cell self-renewal and lineage commitment is critical towards harnessing these cells for therapeutic regeneration. We have previously found that the cell surface ATP binding cassette (ABC)-transporter, Abcg2, influences the proliferation of cardiac side population (CSP) progenitor cells, though through unclear mechanisms. Objective: To determine the role of Abcg2 on cell cycle progression and mode of division in mouse CSP cells. Methods and Results: Herein, using CSP cells isolated from wild-type and Abcg2-knockout mice, we find that Abcg2 regulates G1-S cell cycle transition by FUCCI cell cycle indicators, cell cycle-focused gene expression arrays and confocal live cell fluorescent microscopy. Moreover, we find that modulation of cell cycle results in transition from symmetric to asymmetric cellular division in CSP cells lacking Abcg2. Conclusions: Abcg2 modulates CSP cell cycle progression and asymmetric cell division, establishing a mechanistic link between this surface transporter and cardiac progenitor cell function. Greater understanding of progenitor cell biology, and in particular the regulation of resident progenitor cell homeostasis, is vital for guiding the future development of cell-based therapies for cardiac regeneration.

Abcg2 Regulates Cell Cycle Progression and Asymmetric Division in Mouse Cardiac Side Population Progenitor Cells.

Begr&uuml;ndung: Nach kardialen Sch&auml;digung, kardialen Vorl&auml;uferzellen sind akut reduziert und werden teilweise von geregelten Selbsterneuerung und Verbreitung, die durch symmetrische und asymmetrische Zellteilung Auftritt wieder aufgef&uuml;llt. Verst&auml;ndnis der molekularen Hinweise steuernde Stammvater Zelle Selbsterneuerung und Linie Engagement ist kritisch Nutzbarmachung dieser Zellen f&uuml;r therapeutische Regeneration. Wir haben bereits festgestellt, dass die Zelle Oberfl&auml;che ATP verbindliche Kassette (ABC)-Transporter, Abcg2, beeinflusst die Verbreitung von Progenitorzellen von kardialen Seite Bev&ouml;lkerung (CSP), obwohl durch unklare Mechanismen. Ziel: Die Rolle der Abcg2 auf Zellzyklusprogression und Modus der Division in CSP-Maus bestimmt Zellen. Methoden und Ergebnisse: hierin mit CSP-Zellen isoliert vom Wildtyp und Abcg2-Knockout-M&auml;use, wir finden, dass Abcg2 G1-S Zellzyklus &Uuml;bergang FUCCI Zellzyklus Indikatoren, Zellzyklus-Schwerpunkt Gen-Ausdruck-Arrays und konfokale Leben Lebendzell-Fluoreszenz-Mikroskopie regelt. Dar&uuml;ber hinaus finden wir die Modulation des Zellzyklus Ergebnisse im &Uuml;bergang vom symmetrischen zum asymmetrischen Zellteilung in CSP Zellen Abcg2 fehlt. Fazit: Abcg2 moduliert CSP Zellzyklusprogression und asymmetrische Zellteilung, eine mechanistische Verbindung zwischen dieser Oberfl&auml;che Transporter und kardialen Vorl&auml;uferzellen Zellfunktionen. Besseres Verst&auml;ndnis der Stammvater Zellbiologie und insbesondere die Regulierung der resident Stammvater Zelle Hom&ouml;ostase, ist entscheidend f&uuml;r die zuk&uuml;nftige Entwicklung der zellbasierte Therapien zur kardialen Regeneration F&uuml;hrung.

Abcg2 regelt die Zellzyklusprogression und asymmetrische Aufteilung in Maus kardiale Seite Bev&ouml;lkerung Vorl&auml;uferzellen.

A major hurdle in the use of exogenous stems cells for therapeutic regeneration of injured myocardium remains the poor survival of implanted cells. To date, the delivery of stem cells into myocardium has largely focused on implantation of cell suspensions.

Adult cardiac progenitor cell aggregates exhibit survival benefit both in vitro and in vivo.

Inflammatory cytokines play an important role in the pathogenesis of heart failure. We have recently found the cytokine TWEAK (tumor necrosis factor (TNF)-like weak inducer of apoptosis), a member of the TNF superfamily, to be increased in patients with cardiomyopathy and result in the development of heart failure when overexpressed in mice. The molecular mechanisms underlying TWEAK-induced cardiac pathology, however, remain unknown.

PGC1α plays a critical role in TWEAK-induced cardiac dysfunction.

Immunoglobulin light chain (LC) amyloidosis (AL) results from overproduction of circulating amyloidogenic LC proteins and subsequent amyloid fibril deposition in organs. Mortality in AL amyloidosis patients is highly associated with a rapidly progressive AL cardiomyopathy, marked by profound impairment of diastolic and systolic cardiac function and significant early mortality. While myocardial fibril deposition contributes to the severe diastolic dysfunction seen in AL cardiomyopathy patients, the degree of fibril deposition has not been found to correlate with prognosis. Previously, we and others showed a direct cardiotoxic effect of amyloidogenic LC proteins (AL-LC), which may contribute to the pathophysiology and mortality observed in AL cardiomyopathy patients. However, the mechanisms underlying AL-LC related cardiotoxicity remain unknown. Mammalian stanniocalcin1 (STC1) is associated with a number of cellular processes including oxidative stress and cell death. Herein, we find that STC1 expression is elevated in cardiac tissue from AL cardiomyopathy patients, and is induced in isolated cardiomyocytes in response to AL-LC, but not non-amyloidogenic LC. STC1 overexpression in vitro recapitulates the pathophysiology of AL-LC mediated cardiotoxicity, with increased ROS production, contractile dysfunction and cell death. Overexpression of STC1 in vivo results in significant cardiac dysfunction and cell death. Genetic silencing of STC1 prevents AL-LC induced cardiotoxicity in cardiomyocytes and protects against AL-LC induced cell death and early mortality in zebrafish. The cardiotoxic effects of STC1 appears to be mediated via mitochondrial dysfunction as indicated by loss of mitochondrial membrane potential, ROS production and increased mitochondrial calcium levels. Collectively, this work identifies STC1 as a critical determinant of AL-LC cardiotoxicity.

Stanniocalcin1 is a key mediator of amyloidogenic light chain induced cardiotoxicity.

Microfabrication technology provides a highly versatile platform for engineering hydrogels used in biomedical applications with high-resolution control and injectability. Herein, we present a strategy of microfluidics-assisted fabrication photo-cross-linkable gelatin microgels, coupled with providing protective silica hydrogel layer on the microgel surface to ultimately generate gelatin-silica core-shell microgels for applications as in vitro cell culture platform and injectable tissue constructs. A microfluidic device having flow-focusing channel geometry was utilized to generate droplets containing methacrylated gelatin (GelMA), followed by a photo-cross-linking step to synthesize GelMA microgels. The size of the microgels could easily be controlled by varying the ratio of flow rates of aqueous and oil phases. Then, the GelMA microgels were used as in vitro cell culture platform to grow cardiac side population cells on the microgel surface. The cells readily adhered on the microgel surface and proliferated over time while maintaining high viability (∼90%). The cells on the microgels were also able to migrate to their surrounding area. In addition, the microgels eventually degraded over time. These results demonstrate that cell-seeded GelMA microgels have a great potential as injectable tissue constructs. Furthermore, we demonstrated that coating the cells on GelMA microgels with biocompatible and biodegradable silica hydrogels via sol-gel method provided significant protection against oxidative stress which is often encountered during and after injection into host tissues, and detrimental to the cells. Overall, the microfluidic approach to generate cell-adhesive microgel core, coupled with silica hydrogels as a protective shell, will be highly useful as a cell culture platform to generate a wide range of injectable tissue constructs.

Microfluidics-assisted fabrication of gelatin-silica core-shell microgels for injectable tissue constructs.

AL amyloidosis is the consequence of clonal production of amyloidogenic immunoglobulin light chain (LC) proteins, often resulting in a rapidly progressive and fatal amyloid cardiomyopathy. Recent work has found that amyloidogenic LC directly initiate a cardio-toxic response underlying the pathogenesis of the cardiomyopathy; however, the mechanisms that contribute to this proteotoxicity remain unknown. Using human amyloidogenic LC isolated from patients with amyloid cardiomyopathy, we reveal that dysregulation of autophagic flux is critical for mediating amyloidogenic LC proteotoxicity. Restoration of autophagic flux by pharmacological intervention using rapamycin protected against amyloidogenic light chain protein-induced pathologies including contractile dysfunction and cell death at the cellular and organ level and also prolonged survival in an in vivo zebrafish model of amyloid cardiotoxicity. Mechanistically, we identify impaired lysosomal function to be the major cause of defective autophagy and amyloidogenic LC-induced proteotoxicity. Collectively, these findings detail the downstream molecular mechanisms underlying AL amyloid cardiomyopathy and highlight potential targeting of autophagy and lysosomal dysfunction in patients with amyloid cardiomyopathy.

Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity.

The mechanical properties of the local microenvironment may have important influence on the fate and function of adult tissue progenitor cells, altering the regenerative process. This is particularly critical following a myocardial infarction (MI), in which the normal, compliant myocardial tissue is replaced with fibrotic, stiff scar tissue. In this study, we examined the effects of matrix stiffness on adult cardiac side population (CSP) progenitor cell behavior. Ovine and murine CSP cells were isolated and cultured on polydimethylsiloxane (PDMS) substrates replicating the elastic moduli of normal and fibrotic myocardium. Proliferation capacity and cell cycling was increased in CSP cells cultured on the stiff substrate with an associated reduction in cardiomyogeneic differentiation and accelerated cell ageing. In addition, culture on stiff substrate stimulated upregulation of extracellular matrix (ECM) and adhesion proteins gene expression in CSP cells. Collectively, we demonstrate that microenvironment properties, including matrix stiffness, play a critical role in regulating progenitor cell functions of endogenous resident CSP cells. Understanding the effects of the tissue microenvironment on resident cardiac progenitor cells is a critical step toward achieving functional cardiac regeneration.

ArticleTotal : 2	Year
Publication title Cited by 34	2014
Publication title Cited by 20	2015

Article	Year
Real-time monitoring primary cardiomyocyte adhesion based on electrochemical impedance spectroscopy and electrical cell-substrate impedance sensing. Analytical chemistry\| PubMed ID: 18215019	2008
Impedance-based monitoring of ongoing cardiomyocyte death induced by tumor necrosis factor-alpha. Biophysical journal\| PubMed ID: 19254558	2009
Intervention of cardiomyocyte death based on real-time monitoring of cell adhesion through impedance sensing. Biosensors & bioelectronics\| PubMed ID: 19615884	2009
Intervention of cardiomyocyte death based on the impedance-sensing technique of monitoring cell adhesion. Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference\| PubMed ID: 19964632	2009
Spot identification and quality control in cell-based microarrays. ACS combinatorial science\| PubMed ID: 22850537	2012
Regeneration in heart disease-Is ECM the key? Life sciences\| PubMed ID: 22982346	2012
Abcg2 Regulates Cell Cycle Progression and Asymmetric Division in Mouse Cardiac Side Population Progenitor Cells. Circulation research\| PubMed ID: 23136123	2012
Adult cardiac progenitor cell aggregates exhibit survival benefit both in vitro and in vivo. PloS one\| PubMed ID: 23226295	2012
PGC1α plays a critical role in TWEAK-induced cardiac dysfunction. PloS one\| PubMed ID: 23342071	2013
Stanniocalcin1 is a key mediator of amyloidogenic light chain induced cardiotoxicity. Basic research in cardiology\| PubMed ID: 23982491	2013
Microfluidics-assisted fabrication of gelatin-silica core-shell microgels for injectable tissue constructs. Biomacromolecules\| PubMed ID: 24344625	2014
Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity. EMBO molecular medicine\| PubMed ID: 25319546	2014
A role for matrix stiffness in the regulation of cardiac side population cell function. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 25724498	2015

Yiling Qiu

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