Funding, Deals & Partnerships: BIOLOGICS & MEDICAL DEVICES; BioMed e-Series; Medicine and Life Sciences Scientific Journal – http://PharmaceuticalIntelligence.com
Important but Unseen Human Embryo Developmental Stages Mimicked in Lab
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Scientists have created embryo-like structures that mimic a crucial yet not much known stage of human development. The structures, created from stem cells and called gastruloids, are the first to form a 3D assembly that lays out how the body will take shape. The gastruloids developed rudimentary components of a heart and nervous system, but lacked the components to form a brain and other cell types that would make them capable of becoming a viable fetus.
Human embryos take a momentous leap in their third week, when the largely homogeneous ball of cells starts to differentiate and develop specific characteristics of the body parts they will become, a process known as gastrulation. During this process, the embryo elongates and lays down a body plan with a head and tail, often called the head-to-tail axis. But scientists have never seen this process live in action. That is partly because many countries have regulations that stop embryos from being grown in the laboratory for research beyond 14 days.
Over the past years, several research groups have cultured embryonic stem-cell structures that model when cells start to differentiate. The latest model developed at the University of Cambridge, UK and their collaborators in the Netherlands, Showed for the first time what happens when the blueprint for the body’s development is laid out, around 18–21 days after conception. Genetic analysis showed that the cells formed were those that would eventually go on to form muscles in the trunk, vertebrae, heart and other organs.
If everything is done properly, the cells develop into 3D structures on their own — and then spontaneously mimic the gastrulation process. Although they display certain key features of a 21-day-old embryo, the gastruloids reach that stage after just 72 hours and survive for maximum 4 days before collapsing. Scientists will probably use the model to make structures that are even more realistic representations of early development.
The model could help scientists to understand the role of genetics and environmental factors in different disorders. The artificial structures make it possible to avoid ethical concerns about doing research on human embryos. But as the structures become more advanced and life-like, there may be ethical restrictions.
A heart-healthy diet has been the basis of atherosclerotic cardiovascular disease (ASCVD) prevention and treatment for decades. The potential cardiovascular (CV) benefits of specific individual components of the “food-ome” (defined as the vast array of foods and their constituents) are still incompletely understood, and nutritional science continues to evolve.
The scientific evidence base in nutrition is still to be established properly. It is because of the complex interplay between nutrients and other healthy lifestyle behaviours associated with changes in dietary habits. However, several controversial dietary patterns, foods, and nutrients have received significant media exposure and are stuck by hype.
Decades of research have significantly advanced our understanding of the role of diet in the prevention and treatment of ASCVD. The totality of evidence includes randomized controlled trials (RCTs), cohort studies, case-control studies, and case series / reports as well as systematic reviews and meta-analyses. Although a robust body of evidence from RCTs testing nutritional hypotheses is available, it is not feasible to obtain meaningful RCT data for all diet and health relationships.
Studying preventive diet effects on ASCVD outcomes requires many years because atherosclerosis develops over decades and may be cost-prohibitive for RCTs. Most RCTs are of relatively short duration and have limited sample sizes. Dietary RCTs are also limited by frequent lack of blinding to the intervention and confounding resulting from imperfect diet control (replacing 1 nutrient or food with another affects other aspects of the diet).
In addition, some diet and health relationships cannot be ethically evaluated. For example, it would be unethical to study the effects of certain nutrients (e.g., sodium, trans fat) on cardiovascular disease (CVD) morbidity and mortality because they increase major risk factors for CVD. Epidemiological studies have suggested associations among diet, ASCVD risk factors, and ASCVD events. Prospective cohort studies yield the strongest observational evidence because the measurement of dietary exposure precedes the development of the disease.
However, limitations of prospective observational studies include: imprecise exposure quantification; co-linearity among dietary exposures (e.g., dietary fiber tracks with magnesium and B vitamins); consumer bias, whereby consumption of a food or food category may be associated with non-dietary practices that are difficult to control (e.g., stress, sleep quality); residual confounding (some non-dietary risk factors are not measured); and effect modification (the dietary exposure varies according to individual/genetic characteristics).
It is important to highlight that many healthy nutrition behaviours occur with other healthy lifestyle behaviours (regular physical activity, adequate sleep, no smoking, among others), which may further confound results. Case-control studies are inexpensive, relatively easy to do, and can provide important insight about an association between an exposure and an outcome. However, the major limitation is how the study population is selected or how retrospective data are collected.
In nutrition studies that involve keeping a food diary or collecting food frequency information (i.e., recall or record), accurate memory and recording of food and nutrient intake over prolonged periods can be problematic and subject to error, especially before the diagnosis of disease.
The advent of mobile technology and food diaries may provide opportunities to improve accuracy of recording dietary intake and may lead to more robust evidence. Finally, nutrition science has been further complicated by the influences of funding from the private sector, which may have an influence on nutrition policies and practices.
So, the future health of the global population largely depends on a shift to healthier dietary patterns. Green leafy vegetables and antioxidant suppliments have significant cardio-protective properties when consumed daily. Plant-based proteins are significantly more heart-healthy compared to animal proteins.
However, in the search for the perfect dietary pattern and foods that provide miraculous benefits, consumers are vulnerable to unsubstantiated health benefit claims. As clinicians, it is important to stay abreast of the current scientific evidence to provide meaningful and effective nutrition guidance to patients for ASCVD risk reduction.
Available evidence supports CV benefits of nuts, olive oil and other liquid vegetable oils, plant-based diets and plant-based proteins, green leafy vegetables, and antioxidant-rich foods. Although juicing may be of benefit for individuals who would otherwise not consume adequate amounts of fresh fruits and vegetables, caution must be exercised to avoid excessive calorie intake. Juicing of fruits / vegetables with pulp removal increases calorie intake. Portion control is necessary to avoid weight gain and thus cardiovascular health.
There is currently no evidence to supplement regular intake of antioxidant dietary supplements. Gluten is an issue for those with gluten-related disorders, and it is important to be mindful of this in routine clinical practice; however, there is no evidence for CV or weight loss benefits, apart from the potential caloric restriction associated with a gluten free diet.
Introduction: A just published article from the Gladstone Institute establishes that cardiac muscle can be generated from inducible explandable cardiovascular progenitor cells. However, while the study has validity, it leaves much to be explained, especially in light of the references to many previous studies to generate cardiomycytes for heart failure.
Skin Cells Opening the Door to the Possibility of Personalized Medicine for Heart Attack Patients
Gladstone Institute research scientists have devised a new way to make heart replacement cells. This novel protocol generates cells that lie in between embryonic stem cells and adult heart cells. These induced expandable cardiovascular progenitor cells (ieCPCs) might very well hold the key to treating heart disease. Even though ieCPCs can develop into heart cells, they still have the ability to grow and expand in culture to produce the large numbers of cells required for clinical purposes. When these ieCPCs are injected directly into the hearts of laboratory mice that have recently suffered a heart attack, they formed heart muscle cells and other heart-specific cell types and significantly improved heart function.
Yu Zhang, MD, PhD, lead author on the study and a postdoctoral scholar at the Gladstone Institutes said, “Scientists have tried for decades to treat heart failure by transplanting adult heart cells, but these cells cannot reproduce themselves, and so they do not survive in the damaged heart.” Zhang continued, “Our generated ieCPCs can prolifically replicate and reliably mature into the three types of cells in the heart, which makes them a very promising potential treatment for heart failure.”
CPCs or cardiovascular progenitor cells are the result of embryonic development and help form the embryonic heart. In the embryo, CPCs can differentiate into a wide variety of different heart-specific cells. This Gladstone Institute study, which was published in the journal Cell Stem Cell, Zhang and his colleagues reprogrammed mouse embryonic fibroblasts into CPCs in the laboratory. Once the mouse embryonic fibroblasts had been reprogrammed into CPCs, Zhang and others used a special medium to keep the cells from differentiating into fully-mature, functional heart cells that no longer were able to divide.
CPCs constitute so-called “organ-specific stem cells.” Organ-specific stem cells are special because they can differentiate into adult cells and, under the right conditions, grow, expand and proliferate in culture indefinitely. Zhang and his colleagues were able to expand their ieCPC cultures for over a dozen generations. This generated more than enough cells to treat several patients.
The importance of the ability of these cells to expand in the laboratory cannot be undersold. When a patient suffers a heart attack, over one billion heart cells can die off. Robust cell renewal means ieCPCs can play the role of a sustainable source of cells that can replace the cells that died as a result of the heat attack. Furthermore, ieCPCs can also develop into each of the three different types of heart cells: cardiomyocytes (heart muscle cells), endothelial cells (blood vessel cells), and smooth muscle cells (that surround the blood vessels and regulate their diameter).. When ieCPCs were injected into a mouse hearts, they spontaneously differentiated into each of these heart-specific cell types without requiring any further coaxing or signals.
Previous attempts to treat heart failure by transplanting adult heart cells have produced, for the most part, modest results. Implanted cells tend to survive poorly and do not self-renew, which seriously compromises their ability to repopulate and heal a damaged heart. An additional caveat is that regenerating the heart after a heart attack requires that the heart be supplied with more than just heart muscle cells (cardiomyocytes). Instead the heart needs all three cell types;
Clinical trials that have tested the ability of non-cardiac stem cells to heal the heart after a heart attack have also shown modest, though limited success. In this case, the implanted cells only differentiate into heart-specific cells types rather poorly. Such transdifferentiation events require complex signals that are absent in an adult heart. ieCPCs circumvent these issues since they are already heart-specific progenitor cells that are committed to forming heart-specific cell types.
In this study, 90% of the injected ieCPCs were retained in a mouse heart after a heart attack and successfully differentiated into functioning heart cells. The ieCPCs formed cardiomyocytes that integrated into the myocardium and formed functional connections with existing, surviving cardiomyocytes. The ability to connect with existing heart muscle cells is also crucial to minimize the risk of arrhythmias after a heart attack. The implanted ieCPCs also created new blood vessels that pumped blood and oxygen to newly-forming heart tissues. The ieCPCs significantly improved heart function. The mouse hearts pumped more efficiently, and the benefits lasted for at least three months. Because these cells are generated from skin cells, this procedure also opens the door for personalized medicine in which a heart patient’s own cells are used to treat their heart disease.
ieCPCs Give Rise to CMs, ECs, and SMCs In Vivo and Improve Cardiac Function after MI
(A–E) Immunofluorescence analyses of RFP and CM (A), EC (B and C), and SMC (D and E) markers in tissue sections collected 2 weeks after transplanting RFP-labeled ieCPCs at passage 10 into infarcted hearts of immunodeficient mice. Scale bars represent 100 μm.
(F and G) Ejection fraction and fractional shortening of the left ventricle (LV) quantified by echocardiography. Results from two independent experiments were shown. D, days; W, weeks.
(H–J) Cardiac fibrosis was evaluated at eight levels (L1–L8) by Masson’s trichrome staining 12 weeks after coronary ligation. The ligation site is marked as X. Sections of representative hearts are shown in (I) with quantification in (J). Scar tissue (%) = (the sum of fibrotic area or length at L1–L8/the sum of LV area or circumference at L1–L8) × 100. Scale bars represent 500 μm.
(K) Quantification of LV circumference of mouse hearts 12 weeks after transplantation of 2nd MEFs or ieCPCs. Data were summarized from 48 sections for each group. Data are mean ± SE. ∗p < 0.05.
“Cardiac progenitor cells could be ideal for heart regeneration,” said senior author Sheng Ding, PhD, a senior investigator at Gladstone. “They are the closest precursor to functional heart cells, and, in a single step, they can rapidly and efficiently become heart cells, both in a dish and in a live heart. With our new technology, we can quickly create billions of these cells in a dish and then transplant them into damaged hearts to treat heart failure.”
Discussion: The study raises some important questions.
How are the cultured cells different than those used in previous studies?
Cardiomyocytes and fibroblasts are both of mesodermal origin. What determines which way the stem cell line will differentiate?
What is the difference, if any, between the cell culture environment and the in vivo environment into which they are placed?
There is a difference between chronic hypoxemia with congestive heart failure and acute coronary syndrome. The experiment performed would be more apt to apply to post-ACS than to chronic heart failure.
Functional heart muscle regenerated in decellularized human hearts
A partially recellularized human whole-heart cardiac scaffold, reseeded with human cardiomyocytes derived from induced pluripotent stem cells, being cultured in a bioreactor that delivers a nutrient solution and replicates some of the environmental conditions around a living heart. Credit: Bernhard Jank, MD, Ott Lab, Center for Regenerative Medicine, Massachusetts General Hospital
Massachusetts General Hospital (MGH) researchers have taken some initial steps toward the creation of bioengineered human hearts using donor hearts stripped of components that would generate an immune response and cardiac muscle cells generated from induced pluripotent stem cells (iPSCs), which could come from a potential recipient. The investigators described their accomplishments – which include developing an automated bioreactor system capable of supporting a whole human heart during the recellularization process—earlier this year in Circulation Research.
“Generating functional cardiac tissue involves meeting several challenges,” says Jacques Guyette, PhD, of the MGH Center for Regenerative Medicine (CRM), lead author of the report. “These include providing a structural scaffold that is able to support cardiac function, a supply of specialized cardiac cells, and a supportive environment in which cells can repopulate the scaffold to form mature tissue capable of handling complex cardiac functions.”
The research team is led by Harald Ott, MD, of the MGH CRM and the Department of Surgery, senior author of the paper. In 2008, Ott developed a procedure for stripping the living cells from a donor organ with a detergent solution and then repopulating the remaining extracellular matrix scaffold with organ-appropriate types of cells. Since then his team has used the approach to generate functional rat kidneys and lungs and has decellularized large-animal hearts, lungs and kidneys. This report is the first to conduct a detailed analysis of the matrix scaffold remaining after decellularization of whole human hearts, along with recellularization of the cardiac matrix in three-dimensional and whole-heart formats.
The study included 73 human hearts that had been donated through the New England Organ Bank, determined to be unsuitable for transplantation and recovered under research consent. Using a scaled-up version of the process originally developed in rat hearts, the team decellularized hearts from both brain-dead donors and from those who had undergone cardiac death. Detailed characterization of the remaining cardiac scaffolds confirmed a high retention of matrix proteins and structure free of cardiac cells, the preservation of coronary vascular and microvascular structures, as well as freedom from human leukocyte antigens that could induce rejection. There was little difference between the reactions of organs from the two donor groups to the complex decellularization process.
Instead of using genetic manipulation to generate iPSCs from adult cells, the team used a newer method to reprogram skin cells with messenger RNA factors, which should be both more efficient and less likely to run into regulatory hurdles. They then induced the pluripotent cells to differentiate into cardiac muscle cells or cardiomyocytes, documenting patterns of gene expression that reflected developmental milestones and generating cells in sufficient quantity for possible clinical application. Cardiomyocytes were then reseeded into three-dimensional matrix tissue, first into thin matrix slices and then into 15 mm fibers, which developed into spontaneously contracting tissue after several days in culture.
The last step reflected the first regeneration of human heart muscle from pluripotent stem cells within a cell-free, human whole-heart matrix. The team delivered about 500 million iPSC-derived cardiomyocytes into the left ventricular wall of decellularized hearts. The organs were mounted for 14 days in an automated bioreactor system developed by the MGH team that both perfused the organ with nutrient solution and applied environmental stressors such as ventricular pressure to reproduce conditions within a living heart. Analysis of the regenerated tissue found dense regions of iPSC-derived cells that had the appearance of immature cardiac muscle tissue and demonstrated functional contraction in response to electrical stimulation.
“Regenerating a whole heart is most certainly a long-term goal that is several years away, so we are currently working on engineering a functional myocardial patch that could replace cardiac tissue damaged due a heart attack or heart failure,” says Guyette. “Among the next steps that we are pursuing are improving methods to generate even more cardiac cells – recellularizing a whole heart would take tens of billions—optimizing bioreactor-based culture techniques to improve the maturation and function of engineered cardiac tissue, and electronically integrating regenerated tissue to function within the recipient’s heart.”
Team leader Ott, an assistant professor of Surgery at Harvard Medical School, adds, “Generating personalized functional myocardium from patient-derived cells is an important step towards novel device-engineering strategies and will potentially enable patient-specific disease modeling and therapeutic discovery. Our team is excited to further develop both of these strategies in future projects.”
More information: Jacques P. Guyette et al. Bioengineering Human Myocardium on Native Extracellular MatrixNovelty and Significance, Circulation Research (2016). DOI: 10.1161/CIRCRESAHA.115.306874
Stem cell study in mice offers hope for treating heart attack patients
Cardiac stem cells, pictured here, give hope to patients who have suffered a heart attack. Credit: UCSF
A UCSF stem cell study conducted in mice suggests a novel strategy for treating damaged cardiac tissue in patients following a heart attack. The approach potentially could improve cardiac function, minimize scar size, lead to the development of new blood vessels – and avoid the risk of tissue rejection.
In the investigation, reported online in the journal PLoS ONE, the researchers isolated and characterized a novel type of cardiac stem cell from the heart tissue of middle-aged mice following a heart attack.
Then, in one experiment, they placed the cells in the culture dish and showed they had the ability to differentiate into cardiomyocytes, or “beating heart cells,” as well as endothelial cells and smooth muscle cells, all of which make up the heart.
In another, they made copies, or “clones,” of the cells and engrafted them in the tissue of other mice of the same genetic background who also had experienced heart attacks. The cells induced angiogenesis, or blood vessel growth, or differentiated, or specialized, into endothelial and smooth muscle cells, improving cardiac function.
“These findings are very exciting,” said first author Jianqin Ye, PhD, MD, senior scientist at UCSF’s Translational Cardiac Stem Cell Program. First, “we showed that we can isolate these cells from the heart of middle-aged animals, even after a heart attack.” Second, he said, “we determined that we can return these cells to the animals to induce repair.”
Importantly, the stem cells were identified and isolated in all four chambers of the heart, potentially making it possible to isolate them from patients’ hearts by doing right ventricular biopsies, said Ye. This procedure is “the safest way of obtaining cells from the heart of live patients, and is relatively easy to perform,” he said.
“The finding extends the current knowledge in the field of native cardiac progenitor cell therapy,” said senior author Yerem Yeghiazarians, MD, director of UCSF’s Translational Cardiac Stem Cell Program and an associate professor at the UCSF Division of Cardiology. “Most of the previous research has focused on a different subset of cardiac progenitor cells. These novel cardiac precursor cells appear to have great therapeutic potential.”
The hope, he said, is that patients who have severe heart failure after a heart attack or have cardiomyopathy would be able to be treated with their own cardiac stem cells to improve the overall health and function of the heart. Because the cells would have come from the patients, themselves, there would be no concern of cell rejection after therapy.
The cells, known as Sca-1+ stem enriched in Islet (Isl-1) expressing cardiac precursors, play a major role in cardiac development. Until now, most of the research has focused on a different subset of cardiac progenitor, or early stage, cells known as, c-kit cells.
The Sca-1+ cells, like the c-kit cells, are located within a larger clump of cells called cardiospheres.
The UCSF researchers used special culture techniques and isolated Sca-1+ cells enriched in the Isl-1expressing cells, which are believed to be instrumental in the heart’s development. Since Isl-1 is expressed in the cell nucleus, it has been difficult to isolate them but the new technique enriches for this cell population.
The findings suggest a potential treatment strategy, said Yeghiazarians. “Heart disease, including heart attack and heart failure, is the number one killer in advanced countries. It would be a huge advance if we could decrease repeat hospitalizations, improve the quality of life and increase survival.” More studies are being planned to address these issues in the future.
An estimated 785,000 Americans will have a new heart attack this year, and 470,000 who will have a recurrent attack. Heart disease remains the number one killer in the United States, accounting for one out of every three deaths, according to the American Heart Association.
Medical costs of cardiovascular disease are projected to triple from $272.5 billion to $818.1 billion between now and 2030, according to a report published in the journal Circulation.
Sca-1+ Cardiosphere-Derived Cells Are Enriched for Isl1-Expressing Cardiac Precursors and Improve Cardiac Function after Myocardial Injury
Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.
Methodology/Principal Finding
Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1+CD45– cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1+CD45– cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1+CD45– cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.
Conclusions/Significance
These studies demonstrate that cloned Sca-1+CD45– cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1+) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.
Incorporation of Mg particles into PDLLA regulates mesenchymal stem cell and macrophage responses
Sandra C. Cifuentes1, Fátima Bensiamar2,3, Amparo M. Gallardo-Moreno3,4, Tim A. Osswald5, José L. González-Carrasco1,3, et al.
J Biomed Materials Res Part A 104(4), pages 866–878, April 2016 http://dx.doi.org:/10.1002/jbm.a.35625
Xenotransplantation of Human Cardiomyocyte Progenitor Cells Does Not Improve Cardiac Function in a Porcine Model of Chronic Ischemic Heart Failure. Results f…
Umbilical cord blood cells have an advantage over bone marrow or peripheral blood cells in that aging, systemic inflammation, and stress or damage caused by cell processing procedures can potentially compromise and diminish the regenerative capability of these cells. This problem is particularly acute in the case of treating patients who have recently suffered a heart attack, since transplanted cells experience a rather hostile environment that kills off most cells. Additionally, blood flow through the heart tends to wash out infused cells, which further decreases any regenerative activities the cells might have otherwise exerted.
With this in mind, Patrick Hsieh and his colleagues at the Academia Sinica, in Taipei, Taiwan tested if ability of human cord blood mononuclear cells (CB-MNCs) injected into the heart in combination with a hyaluronan (HA) hydrogel could extend the regenerative abilities of these cells in a pig model. HA is a common component of connective tissue, and, in general, it is very well tolerated by patients and implanted cells. Furthermore, it has the added bonus of shielding cells from a hostile environment and preventing them from being washed out of the heart.
Hsieh used a total of 34 minipigs and divided them into five different groups. One group was the sham operation group in which minipigs received surgical incisions but no heart attack was induced. The second group had heart attacks surgically induced and received infusions of normal saline solutions. The third group of minipigs also experienced heart attacks, and had HA injected into the heart walls. The fourth group also suffered heart attacks and received injections of human umbilical cord stem cells into their heart walls. The fifth group experienced heart attacks and received injections of both HA and human umbilical cord blood cells. The animals were kept and examined two months after surgery.
Two months after the surgery, the minipigs that received injections of human umbilical cord blood cells plus HA showed the highest left ventricle ejection fraction (51.32% ± 0.81%). This is significant when compared to 42.87% ± 0.97%, for the group that received injections of normal saline, 44.2% ± 0.63% for the group that received injections of HA alone, and 46.17% ± 0.39% for the group that received injections of umbilical cord blood cells only. Additionally, hearts from minipigs that received cord blood cells plus HA improved the systolic and diastolic function significantly better than the other experimental groups. Injections of either cord blood cells alone or in combination with HA significantly decreased the scar area and promoted the formation of new blood vessels in the infarcted region. In general, this study suggests that combined infusion of umbilical cord blood cells and HA improves the function of the heart after a heart attack and might prove to be a promising treatment option of heart attack patients.
This is a preclinical study, but it is a preclinical study in a larger animal model system. Umbilical cord blood cells have a demonstrated ability to induce healing in the heart after a heart attack. However, the combination of these cells with HA almost certainly significantly increases cell retention in the heart, thereby significantly improving cardiac performance, and preventing cardiac remodeling. Therefore, using healthy cells donated from another source to replace damaged or moribund cells may be a better option to treat a heart patient and repair their sick heart.
Enabling Technologies for Cell-Based Clinical Translation:Injection of Human Cord Blood Cells With Hyaluronan Improves Postinfarction Cardiac Repair in Pigs
Ming-Yao Chang, Tzu-Ting Huang, Chien-Hsi Chen, Bill Cheng, Shiaw-Min Hwang, Patrick C.H. Hsieh
Stem Cells Trans Med first published on November 16, 2015;doi:10.5966/sctm.2015-0092
Injection of Human Cord Blood Cells With Hyaluronan Improves Postinfarction Cardiac Repair in Pigs
Although safe, recent clinical trials using autologous bone marrow or peripheral blood cells to treat myocardial infarction (MI) show controversial results. These discrepancies are likely caused by factors such as aging, systemic inflammation, and cell processing procedures, all of which might impair the regenerative capability of the cells used. Here, we tested whether injection of human cord blood mononuclear cells (CB-MNCs) combined with hyaluronan (HA) hydrogel improves cell therapy efficacy in a pig MI model. A total of 34 minipigs were divided into 5 groups: sham operation (Sham), surgically induced-MI plus injection with normal saline (MI+NS), HA only (MI+HA), CB-MNC only (MI+CB-MNC), or CB-MNC combined with HA (MI+CB-MNC/HA). Two months after the surgery, injection of MI+CB-MNC/HA showed the highest left ventricle ejection fraction (51.32% ± 0.81%) compared with MI+NS (42.87% ± 0.97%, p < .001), MI+HA (44.2% ± 0.63%, p < .001), and MI+CB-MNC (46.17% ± 0.39%, p < .001) groups. The hemodynamics data showed that MI+CB-MNC/HA improved the systolic function (+dp/dt) and diastolic function (−dp/dt) as opposed to the other experimental groups, of which the CB-MNC alone group only modestly improved the systolic function (+dp/dt). In addition, CB-MNC alone or combined with HA injection significantly decreased the scar area and promoted angiogenesis in the infarcted region. Together, these results indicate that combined CB-MNC and HA treatment improves heart performance and may be a promising treatment for ischemic heart diseases.
Significance
This study using healthy human cord blood mononuclear cells (CB-MNCs) to treat myocardial infarction provides preclinical evidence that combined injection of hyaluronan and human CB-MNCs after myocardial infarction significantly increases cell retention in the peri-infarct area, improves cardiac performance, and prevents cardiac remodeling. Moreover, using healthy cells to replace dysfunctional autologous cells may constitute a better strategy to achieve heart repair and regeneration.
The Mount Sinai researchers believe Cdx2 placental cells offer several important advantages over other types of cells that have been studied in cardiovascular disorders. They not only express proteins that have the ability to generate all the organs in the body, they also have proteins that allow them to travel to injury sites. Plus, they don’t seem to cause a damaging immune response, they reported.
The team was able to isolate Cdx2 cells from full-term human placentas, too, raising the possibility of being able to harvest the treatment from an almost “limitless source” of placentas that would normally be discarded, said principal investigator Hina Chaudhry, M.D., director of cardiovascular regenerative medicine at the Icahn School, in a statement.
“These findings may also pave the way to regenerative therapy of other organs besides the heart,” Chaudhry added.
Cells in the heart expressing the marker cKit were once thought to be the key to cardiac regeneration. These cardiac precursors, researchers found, could proliferate—opening up the opportunity for a way to regrow an organ that until this century was thought incapable of regeneration.
But even as positive results shook out of an early stage clinical trial, a shadow moved in over cKit+ cells, with several labs producing data questioning their reparative powers. Skepticism culminated with a report in 2014 showing that cKit+ cells in mice very rarely produce new heart muscle cells, or cardiomyocytes. The story of cKit+ cells, said Joshua Hare of the University of Miami Miller School of Medicine, “is a very controversial one.”
In the latest development in the cKit+ saga, published this month (October 5) in PNAS, Hare’s team found that cKit+ cells readily become cardiac muscle cells in vitro, as long as the right cellular conditions are present. This could perhaps explain why other groups haven’t seen cKit+ cells becoming cardiomyocytes in vivo that often, he said. “It’s not that the cells don’t have the capacity [to differentiate], but they’re entering the heart at a time that’s nonpermissive for them to become cardiac myocytes.”
Specifically, the researchers found that if they interfered with bone morphogenetic protein signaling—crucial during the development of the heart and other tissues—mouse induced pluripotent stem cells (iPSCs) expressing KIT would become cardiomyocytes. They also demonstrated with genetic fate-mapping that cKit+ cells derive from the neural crest during development and are present in the mouse embryonic heart.
Hare’s group did not find that cKit+ cells have a high propensity to become endothelium, as did the aforementioned 2014 study, which also used genetic fate-mapping. Jeffery Molkentin of Cincinnati Children’s Hospital Medical Center who led that work declined to be interviewed for this story. Hare said the discrepancy could be due to the teams’ different genetic constructs.
Bernardo Nadal-Ginard, an honorary professor at King’s College London whose work has supported the myogenic capacity of cKit+ cells, said he found the evidence from Hare showing they can become myocytes “convincing.” However, he added, “the paper claims the quandary and the dispute is over. But, unfortunately, it is not.”
The paper is more qualitative than quantitative, said Nadal-Ginard, meaning researchers still don’t know how often cKit+ cells become myocytes and whether they become other types of cells (and at what frequency).
Michael Kotlikoff of Cornell University pointed out that Hare’s team didn’t demonstrate that cKit+ cells in vivo have the same regenerative capacity as the iPSCs in vitro. “They never show the myogenic potential of those cells and don’t show them giving rise to cardiomyogensis” in vivo, Kotlikoff told The Scientist. “The expression of [cKit], per se, is not sufficient to identify cells as precursors and the further presumption that signaling processes observed in in vitro differentiation experiments limit such cells from undergoing myogenesis in the adult heart, the stage at which clinical regenerative efforts are focussed, is not supported by data,” he added in an email.
Hare is involved in two planned clinical trials that will administer cKit+ cells to patients with heart failure. (He founded a company called Vestion that is developing cardiac cell therapies.) Already, a phase 1 trial called SCIPIO, which Hare was not part of, found positive signs of tissue repair among patients given their own cKit+ cells. But as questions were raised about the regenerative abilities of these cells, some advocated to wait on the clinical trials until the biology was worked out. Hare said his study does not explain SCIPIO’s results; rather, it offers some clues as to how researchers can boost the reparative potential of these cells.
“To say human trials should be stopped because the experiment didn’t work in the mouse is a bit aggressive,” said Brigham and Women’s Hospital’s Piero Anversa, a leading proponent of cKit+ cells who was involved in SCIPIO and who also found Hare’s results convincing. (Anversa’s own work in the field has been a source of controversy, with an expression of concern issued about some SCIPIO results.) “The answer is going to be in the trial. If the trial goes well we win, if the trial doesn’t go well, we lose.”
K.E. Hatzistergos et al., “cKit+ cardiac progenitors of neural crest origin,” PNAS, 112:13051-56, 2015.
More Doubt Cast Over Cardiac Stem Cells
Contrary to previous reports, cell lineage tracing reveals stem cells in the heart rarely contribute to new muscle.
By Kerry Grens | May 7, 2014
http://www.the-scientist.com/?articles.view/articleNo/39912/title/More-Doubt-Cast-Over-Cardiac-Stem-Cells/
FLICKR, GEORGE SHULKINC-kit cells, which are found in the heart and supposedly act as cardiac stem cells, are the basis of a clinical trial to repair cardiac injury. But a new study published in Nature today (May 7) adds what some researchers are calling “definitive” evidence to the idea that these cells hardly ever produce new heart muscle in vivo. Using genetic lineage tracing in a mouse, a team led by Jeff Molkentin of Cincinnati Children’s Hospital Medical Center found that, while c-kit cells readily produce cardiac endothelium, they very rarely generate cardiomyocytes.
“The conclusion I am led to from this is that the c-kit cell is not a cardiac stem cell, at least in term of its normal, in vivo role,” said Charles Murry, a heart regeneration researcher at the University of Washington who was not involved in this study.
The latest findings add to a string of recent setbacks for advancing the use of these cells as a therapy—including a retraction and an expression of concern regarding two publications and an institutional investigation of one of the leaders in the field, Piero Anversa at Harvard Medical School. “There’s been a tidal wave in the last few weeks of rising skepticism,” said Eduardo Marbán, an author of the new study and a cardiologist at the Cedars-Sinai Heart Institute in Los Angeles. Still, he said, the dispute is not settled, and many stand by the regenerative power of these cells.
“Unequivocal” results
Research led by Anversa has shown that c-kit cells—cardiac progenitor cells expressing the cell surface protein c-kit—can produce new cardiomyocytes. Anversa and others have helped usher the cells into clinical trials to test whether they might help repair damaged cardiac tissue.
Work by other teams, however, has raised doubts about the potential for c-kit cells to actually build new heart muscle. To help resolve the discrepancy, Molkentin and his colleagues developed a mouse in which any cell expressing c-kit—and any of that cell’s progeny—would glow green by a green fluorescent protein tagged to the Kit locus. They found that just 0.027 percent of the myocytes in the mouse heart originated from c-kit cells. “C-kit cells in the heart don’t like to make myocytes,” Molkentin told The Scientist. “We’re not saying anything that’s different” from groups that have not had success with c-kit cells in the past, Molkentin said, “we’re just saying we did it in a way that’s unequivocal.”
Molkentin’s study did not address why there’s a discrepancy between his results and those of Anversa and another leader in the c-kit field, Bernardo Nadal-Ginard, an honorary professor at King’s College London. Last year, Nadal-Ginard and his colleagues showed in Cell that heart regeneration in rodents relies on c-kit positive cells and that depleting these cells abolishes the heart’s ability to repair itself. Nadal-Ginard toldThe Scientist that technical issues with Molkentin’s mouse model could have affected his results, causing too few c-kit cells to be labeled. Additionally, “the work presented by Molkentin used none of our experimental approaches; therefore, it is not possible to compare the results,” Nadal-Ginard said in an e-mail.
In an e-mail to The Scientist, Anversa said his lab is working with the same mouse model Molkentin used, “but our data are too preliminary to make any specific comment. Time will tell.”
Clinical future
Molkentin’s paper only serves to darken the cloud that has moved over Anversa’s work on c-kit cells. Last month, a 2012 paper in Circulation by Anversa’s team was retracted because the data were “sufficiently compromised.” Days later, The Lancet published an expression of concern regarding supplemental data in the published results from the human clinical trial using autologous c-kit cells. Harvard Medical School and Brigham and Women’s Hospital continue to investigate what may have gone wrong.
Meanwhile, Marbán is advancing another type of stem cell, called cardiosphere-derived cells, through human clinical trials to try and treat tissue damage after a heart attack. Marbán said he had been a true believer in c-kit cells, until the data started mounting against them. “The totality of the evidence now says the c-kit cell is no longer a cardiomyocyte progenitor,” he told The Scientist.
If c-kit cells don’t produce new cardiomyocytes, as Molkentin and Marbán assert, where does that leave the clinical trial? Murry said that just because the preclinical, mechanistic basis for the human study is foundering, any promising clinical results are not to be dismissed. “Those results can be considered independent,” he said. Molkentin said it’s possible that c-kit cells work in unknown ways to repair heart tissue. He noted that clinical treatment involves high levels of c-kit cells immersed in culture conditions. “Perhaps these cells act a little different,” Molkentin said.
Nadal-Ginard did not dispute that discrepancies exist between his data and those of others, and agreed that these differences ought to be addressed. He said he’d be willing to work with Molkentin to get to the bottom of it. “The concept under dispute is too important for the field of regenerative medicine—and regenerative cardiology, in particular—to turn into a philosophical/dogmatic argument instead of settling it in a proper scientific manner.”
J.H. van Berlo et al., “c-kit1 cells minimally contribute cardiomyocytes to the heart,” Nature, doi:10.1038/nature13309, 2014.
A high-resolution genetic lineage-tracing study in mice reveals that cKit identifies multipotent progenitors of cardiac neural crest (CNC) origin. Normally, the proportion of cardiomyocytes produced from this lineage is limited, not because of poor differentiation capacity as previously thought, but because of stage-specific changes in the activity of the bone morphogenetic protein pathway. Transient bone morphogenetic protein antagonism efficiently directs mouse iPSCs toward the CNC lineage and, consequently, the generation of cKit+ CNCs with full capacity to form cardiomyocytes and other CNC derivatives in vitro. These findings resolve a long-standing controversy regarding the role of cKit in the heart, and are expected to lead to the development of novel stem cell-based therapies for the prevention and treatment of cardiovascular disease.
Abstract
The degree to which cKit-expressing progenitors generate cardiomyocytes in the heart is controversial. Genetic fate-mapping studies suggest minimal contribution; however, whether or not minimal contribution reflects minimal cardiomyogenic capacity is unclear because the embryonic origin and role in cardiogenesis of these progenitors remain elusive. Using high-resolution genetic fate-mapping approaches withcKitCreERT2/+ and Wnt1::Flpe mouse lines, we show that cKit delineates cardiac neural crest progenitors (CNCkit). CNCkit possess full cardiomyogenic capacity and contribute to all CNC derivatives, including cardiac conduction system cells. Furthermore, by modeling cardiogenesis in cKitCreERT2-induced pluripotent stem cells, we show that, paradoxically, the cardiogenic fate of CNCkit is regulated by bone morphogenetic protein antagonism, a signaling pathway activated transiently during establishment of the cardiac crescent, and extinguished from the heart before CNC invasion. Together, these findings elucidate the origin of cKit+ cardiac progenitors and suggest that a nonpermissive cardiac milieu, rather than minimal cardiomyogenic capacity, controls the degree of CNCkit contribution to myocardium.
This e-Book is a comprehensive review of recent Original Research on METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases. It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences. Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation.The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012. All new articles on this subject, will continue to be incorporated, as published with periodical updates.
We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.
All forthcoming BioMed e-Book Titles can be viewed at:
Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.
Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:
delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
providing a social platform for scientists and clinicians to enter into discussion using social media
compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform
This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.
Table of Contents forMetabolic Genomics & Pharmaceutics, Vol. I
Chapter 1: Metabolic Pathways
Chapter 2: Lipid Metabolism
Chapter 3: Cell Signaling
Chapter 4: Protein Synthesis and Degradation
Chapter 5: Sub-cellular Structure
Chapter 6: Proteomics
Chapter 7: Metabolomics
Chapter 8: Impairments in Pathological States: Endocrine Disorders; Stress
Hypermetabolism and Cancer
Chapter 9: Genomic Expression in Health and Disease
Optogenetics: The Promise for development of Biological Alternatives to the Electronic Pacemaker: Pacing and Resynchronizing Heartbeat by Activating Light-sensitive Proteins: ion-channel ChR2, overexpressed in Excitable cells in Heart Muscle Cells to modulate their Electrical Activity
Reporter: Aviva Lev-Ari, PhD, RN
Optogenetics for in vivo cardiac pacing and resynchronization therapies
Abnormalities in the specialized cardiac conduction system may result in slow heart rate or mechanical dyssynchrony. Here we apply optogenetics, widely used to modulate neuronal excitability1, 2, 3, 4, for cardiac pacing and resynchronization. We used adeno-associated virus (AAV) 9 to express the Channelrhodopsin-2 (ChR2) transgene at one or more ventricular sites in rats. This allowed optogenetic pacing of the hearts at different beating frequencies with blue-light illumination both in vivo and in isolated perfused hearts. Optical mapping confirmed that the source of the new pacemaker activity was the site of ChR2 transgene delivery. Notably, diffuse illumination of hearts where the ChR2 transgene was delivered to several ventricular sites resulted in electrical synchronization and significant shortening of ventricular activation times. These findings highlight the unique potential of optogenetics for cardiac pacing and resynchronization therapies.
The study was conducted by Dr. Udi Nussinovitch as part of his PhD work in Professor Gepstein’s laboratory at the Technion. Dr. Nussinovitch is currently an intern at the Department of Internal Medicine at Rambam.
The optogenetic technology employed allowed researchers to selectively activate light-sensitive proteins (such as the ion-channel ChR2, first identified in algae), which were overexpressed in excitable cells (such as nerve or muscle cells), in an attempt to modulate (either augment or suppress) their electrical activity. Optogenetics has become an important tool in brain research and the current study is the first to translate this important innovation to pace and resynchronize the heartbeat.
In the study, conducted in rats, the researchers first directed a beam of blue light at an area in the heart where the light-sensitive genes were delivered. This resulted in effective pacing of the heart at different rates as dictated by the frequency of the blue light flashes applied. Subsequently, a more advanced experiment was conducted, in which various locations in the rat hearts expressing ChR2 were activated simultaneously by light, resulting in improved synchronization of the contractions of the ventricles.
Professor Gepstein stresses that this is a preliminary study, and that “in order to translate the aforementioned approach to the clinical arena, we must overcome some significant hurdles. We must
improve the penetration of light through the tissues,
ensure continuous expression of the protein in the heart for many years, and
develop a unique pacing device that will provide the necessary illumination.
But despite all of this, the results of the study demonstrate the unique potential of optogenetics for both
cardiac pacing (as an alternative to electronic pacemakers) and
resynchronization (for the treatment of heart failure with ventricular dys-synchrony) therapies.”
Obesity associated with reduced posterior LA endocardial voltage and infiltration of contiguous posterior LA muscle by epicardial fat, representing a unique substrate for atrial fibrillation (AF)
In the imtroduction to this series of discussions I pointed out JEDS Rosalino’s observation about the construction of a complex molecule of acetyl coenzyme A, and the amount of genetic coding that had to go into it. Furthermore, he observes – Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.
acetylCoA
In the tutorial that follows we find support for the view that mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, are achieving a separation from inconsistent views introduced by the classical model of molecular biology and genomics, toward a more functional cellular dynamics that is not dependent on the classic view. The classical view fits a rigid framework that is to genomics and metabolomics as Mendelian genetics if to multidimentional, multifactorial genetics. The inherent difficulty lies in two places:
Interactions between differently weighted determinants
A large part of the genome is concerned with regulatory function, not expression of the code
The goal of the tutorial was to achieve an understanding of how cell signaling occurs in a cell. Completion of the tutorial would provide
a basic understanding signal transduction and
the role of phosphorylation in signal transduction.
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin
In addition – detailed knowledge of –
the role of Tyrosine kinases and
G protein-coupled receptors in cell signaling.
serine
threonine
protein kinase
We are constantly receiving and interpreting signals from our environment, which can come
in the form of light, heat, odors, touch or sound.
The cells of our bodies are also
constantly receiving signals from other cells.
These signals are important to
keep cells alive and functioning as well as
to stimulate important events such as
cell division and differentiation.
Signals are most often chemicals that can be found
in the extracellular fluid around cells.
These chemicals can come
from distant locations in the body (endocrine signaling by hormones), from
nearby cells (paracrine signaling) or can even
be secreted by the same cell (autocrine signaling).
Signaling molecules may trigger any number of cellular responses, including
changing the metabolism of the cell receiving the signal or
result in a change in gene expression (transcription) within the nucleus of the cell or both.
controlling the output of ribosomes.
To which I would now add..
result in either an inhibitory or a stimulatory effect
The three stages of cell signaling are:
Cell signaling can be divided into 3 stages:
Reception: A cell detects a signaling molecule from the outside of the cell.
Transduction: When the signaling molecule binds the receptor it changes the receptor protein in some way. This change initiates the process of transduction. Signal transduction is usually a pathway of several steps. Each relay molecule in the signal transduction pathway changes the next molecule in the pathway.
Response: Finally, the signal triggers a specific cellular response.
Signal Transduction – ligand binds to surface receptor
Membrane receptors function by binding the signal molecule (ligand) and causing the production of a second signal (also known as a second messenger) that then causes a cellular response. These types of receptors transmit information from the extracellular environment to the inside of the cell.
by changing shape or
by joining with another protein
once a specific ligand binds to it.
Examples of membrane receptors include
G Protein-Coupled Receptors and
Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.
Intracellular receptors are found inside the cell, either in the cytopolasm or in the nucleus of the target cell (the cell receiving the signal).
Note that though change in gene expression is stated, the change in gene expression does not here imply a change in the genetic information – such as – mutation. That does not have to be the case in the normal homeostatic case.
This point is the differentiating case between what JEDS Roselino has referred as
a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
a one-to-one transcription of the genetic code.
The rate of transcription can be controlled, or it can be blocked. This is in large part in response to the metabolites in the immediate interstitium.
This might only be
a change in the rate of a transcription or a suppression of expression through RNA.
Or through a conformational change in an enzyme
Swinging domains in HECT E3 enzymes
Since signaling systems need to be
responsive to small concentrations of chemical signals and act quickly,
cells often use a multi-step pathway that transmits the signal quickly,
while amplifying the signal to numerous molecules at each step.
Signal transduction pathways are shown (simplified):
Signal Transduction
Signal transduction occurs when an
extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.
In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell. For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.
Guanosine monophosphate structure
In 2007, a total of 48,377 scientific papers—including 11,211 e-review papers—were published on the subject. The term first appeared in a paper’s title in 1979. Widespread use of the term has been traced to a 1980 review article by Rodbell: Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.
Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation.
This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.
Integrin
Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.
steroid hormone receptor
Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye, and odorants binding to odorant receptors in the nasal epithelium. Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.
Signal transduction cascades amplify the signal output
Signal transduction cascades amplify the signal output
G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling
signal transduction pathways
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling
Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ. Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits.
The dissociation exposes sites on the subunits that can interact with other molecules. The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.
Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.
insulin receptor and and insulin receptor signaling pathway (IRS)
To perform signal transduction, RTKs need to form dimers in the plasma membrane; the dimer is stabilized by ligands binding to the receptor.
RTKs
The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.
Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.
Signal-Transduction-Pathway
As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are
members of the Ras, Rho, and Raf families, referred to collectively as small G proteins.
They act as molecular switches usually
tethered to membranes by isoprenyl groups linked to their carboxyl ends.
Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate
small G proteins that activate guanine nucleotide exchange factors such as SOS1.
Once activated, these exchange factors can activate more small G proteins, thus
amplifying the receptor’s initial signal.
The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.
Integrin
Integrin
Integrin-mediated signal transduction
An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).
Integrins are produced by a wide variety of cells; they play a role in
cell attachment to other cells and the extracellular matrix and
in the transduction of signals from extracellular matrix components such as fibronectin and collagen.
Ligand binding to the extracellular domain of integrins
changes the protein’s conformation,
clustering it at the cell membrane to
initiate signal transduction.
Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.
As shown in the picture, cooperative integrin-RTK signaling determines the
timing of cellular survival,
apoptosis,
proliferation, and
differentiation.
integrin-mediated signal transduction
Integrin signaling
ion channel
A ligand-gated ion channel, upon binding with a ligand, changes conformation
to open a channel in the cell membrane
through which ions relaying signals can pass.
An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels
induces action potentials, such as those that travel along nerves,
by depolarizing the membrane of post-synaptic cells,
resulting in the opening of voltage-gated ion channels.
RyR and Ca+ release from SR
An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+;
it acts as a second messenger
initiating signal transduction cascades and
altering the physiology of the responding cell.
This results in amplification of the synapse response between synaptic cells
by remodelling the dendritic spines involved in the synapse.
In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3,
cAMP
Inositol_1,4,5-trisphosphate.svg
the latter controlling the release of intracellular calcium stores into the cytoplasm.
Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example,
calcium ions bind to the EF hand domains of calmodulin,
allowing it to bind and activate calmodulin-dependent kinase.
calcium movement and RyR2 receptor
PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.
Signals can be generated within organelles, such as chloroplasts and mitochondria, modulating the nuclear
gene expression in a process called retrograde signaling.
Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data of Arabidopsis thaliana, revealed the identification of metabolites which are putatively acting as mediators of nuclear gene expression.
Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).
Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,
DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.
DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.
On the other hand,
zinc is the most abundant trace metal in the human brain.
There are many scientific studies linking zinc, especially
excess amounts of free zinc, to cellular death.
Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between
omega-3 fatty acids, zinc transporter levels and
free zinc availability at cellular levels.
Many other studies have also suggested a possible
omega-3 and zinc effect on neurodegeneration and cellular death.
Therefore, in this review, we will examine
the effect of omega-3 fatty acids on zinc transporters and
the importance of free zinc for human neuronal cells.
Moreover, we will evaluate the collective understanding of
mechanism(s) for the interaction of these elements in neuronal research and their
significance for the diagnosis and treatment of neurodegeneration.
Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to
reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in
Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as
cellular differentiation,
photoreceptor membrane biogenesis and
active synaptogenesis.
Despite the significance of these
two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both
omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
opposing physiological effects. In the human body,
high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
thereby increase inflammatory processes [10].
the reverse process can be seen with increased omega-3 fatty acids in the body.
Many other factors, such as
thromboxane A2 (TXA2),
leukotriene
B4 (LTB4),
IL-1,
IL-6,
tumor necrosis factor (TNF) and
C-reactive protein,
which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.
Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in
immune responses,
gene expression and
intercellular communications.
omega-3 fatty acids are known to be vital in
the prevention of fatal ventricular arrhythmias, and
are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels
.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.
The main
In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that
only high fish consumption, but
not dietary omega-3 acid intake,
had a protective effect on cognitive decline
In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown
a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.
A trial of subjects with mild memory complaints demonstrated
an improvement with 900 mg of DHA.
We review key findings on
the effect of the omega-3 fatty acid DHA on zinc transporters and the
importance of free zinc to human neuronal cells.
DHA is the most abundant fatty acid in neural membranes, imparting appropriate
fluidity and other properties,
and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated
in membrane phospholipids at synapses and
in retinal photoreceptors and
also in the testis and sperm.
In adult rats’ brain, DHA comprises approximately
17% of the total fatty acid weight, and
in the retina it is as high as 33%.
DHA is believed to have played a major role in the evolution of the modern human –
in particular the well-developed brain.
Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.
Analysis of human cadaver brains have shown that
people with AD have less DHA in their frontal lobe
and hippocampus compared with unaffected individuals
Furthermore, studies in mice have increased support for the
protective role of omega-3 fatty acids.
Mice administrated with a dietary intake of DHA showed
an increase in DHA levels in the hippocampus.
Errors in memory were decreased in these mice and they demonstrated
reduced peroxide and free radical levels,
suggesting a role in antioxidant defense.
Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary
DHA supplementation had a protective effect against reduction in
drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
decreased caspase activity.
Zinc
Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to
growth,
development,
differentiation,
immune response,
receptor activity,
DNA synthesis,
gene expression,
neuro-transmission,
enzymatic catalysis,
hormonal storage and release,
tissue repair,
memory,
the visual process
and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects, as
a component of over 300 different enzymes
an integral component of a metallothioneins
a gene regulatory protein.
Approximately 3% of all proteins contain
zinc binding motifs .
The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;
catalytic,
coactive and
Indeed, it is the only metal found in all six different subclasses
of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.
Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated
through glutamate receptors, and
it inhibits excitatory and inhibitory receptors.
Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and
its release is dependent on neural activity.
Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.
The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to
form insoluble amyloid in the presence of
high physiological levels of zinc.
Insoluble amyloid is thought to
aggregate to form plaques,
which is a main pathological feature of AD. Further studies have shown that
chelation of zinc ions can deform and disaggregate plaques.
In AD, the most prominent injuries are found in
hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
somatostatin-containing neurons in the forebrain.
All of these neurons are known to favor
rapid and direct entry of zinc in high concentration
leaving neurons frequently exposed to high dosages of zinc.
This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of
inhibiting Ca2+ and Na+ voltage gated channels
and up-regulating the cellular levels of reactive oxygen species (ROS).
High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.
If the human diet is deficient in zinc, the body
efficiently conserves zinc at the tissue level by compensating other cellular mechanisms
to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and
redistribution of available zinc to more zinc dependent cells or organs.
A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the
assessment of the zinc status of individuals and populations.
In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting
as a target for zinc ion binding and thus
assisting in the trafficking of zinc ions through the cell,
which may be similar to that of zinc transporters
Zinc Transporters
Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur
via intermembrane proteins and zinc transporter (ZnT) proteins
These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).
Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).
The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where
it is responsible for the transport of zinc into the synaptic vesicles of
glutamatergic neurons in the hippocampus and neocortex,
Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/. http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg
Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].
omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA),
docosahexenoic acid (DHA) and
eicosapentaenoic acid (EPA).
In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between
increased fish consumption and reduced risk of AD
and eight out of ten studies have reported a link between higher blood omega-3 levels
DHA and Zinc Homeostasis
Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in
increased zinc levels in the hippocampus and
elevated expression of the putative zinc transporter, ZnT3, in the rat brain.
Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,
a significant reduction in plaque load
and the presence of insoluble amyloid were observed.
In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant
reduction in free zinc availability in the hippocampus
and cerebral cortex.
Collectively, the findings from this study are very interesting and indicate a clear connection between
zinc availability and amyloid plaque formation,
thus indicating a possible link to AD.
DHA supplementation has also been reported to limit the following:
amyloid presence,
synaptic marker loss,
hyper-phosphorylation of Tau,
oxidative damage and
cognitive deficits in transgenic mouse model of AD.
In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.
In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.
When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible
neuroprotective mechanism of DHA,
which we believe is exerted through
a reduction in cellular zinc levels (through altering zinc transporter expression levels)
that in turn inhibits apoptosis.
DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and
free zinc levels in the cytosol were significantly low compared to the control
This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had
low levels of active caspase-3 and
high Bcl-2 levels compared to the control treatment.
These findings are consistent with previous published data and further strengthen the possible
correlation between zinc, DHA and neurodegeneration.
On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of
ZnT3 in memory and AD pathology.
For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of
ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
required for hippocampus-dependent memory
Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.
2012pharmaceutical
Amandeep Kaur
Aashir Awan, Phd
Abhisar Anand
Adina Hazan
Alan F. Kaul, PharmD., MS, MBA, FCCP
alexcrystal6
anamikasarkar
apreconasia
aviralvatsa
David Orchard-Webb, PhD
danutdaagmailcom
Demet Sag, Ph.D., CRA, GCP
Dror Nir
dsmolyar
Ethan Coomber
evelinacohn
FrasonFrancis
Gail S Thornton
Irina Robu
jayzmit48
jdpmd
jshertok
kellyperlman
Ed Kislauskis
larryhbern
Madison Davis
marzankhan
megbaker58
ofermar2020
Dr. Pati
pkandala
ritusaxena
Rick Mandahl
sjwilliamspa
Srinivas Sriram
stuartlpbi
Dr. Sudipta Saha
tildabarliya
zraviv06
zs22
