Prabhakar N.R.,University of Chicago |
Semenza G.L.,Institute for Cell Engineering
Pflugers Archiv European Journal of Physiology | Year: 2016
Oxygen (O2) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Carotid body responses to hypoxia are not uniform but instead exhibit remarkable inter-individual variations. The molecular mechanisms underlying variations in carotid body O2 sensing are not known. Hypoxia-inducible factor-1 (HIF-1) and HIF-2 mediate transcriptional responses to hypoxia. This article reviews the emerging evidence that proper expression of the HIF-α isoforms is a key molecular determinant for carotid body O2 sensing. HIF-1α deficiency leads to a blunted carotid body hypoxic response, which is due to increased abundance of HIF-2α, elevated anti-oxidant enzyme activity, and a reduced intracellular redox state. Conversely, HIF-2α deficiency results in augmented carotid body sensitivity to hypoxia, which is due to increased abundance of HIF-1α, elevated pro-oxidant enzyme activity, and an oxidized intracellular redox state. Double heterozygous mice with equally reduced HIF-1α and HIF-2α showed no abnormality in redox state or carotid body O2 sensing. Thus, mutual antagonism between HIF-α isoforms determines the redox state and thereby establishes the set point for hypoxic sensing by the carotid body. © 2015, Springer-Verlag Berlin Heidelberg.
Semenza G.L.,Institute for Cell Engineering |
Semenza G.L.,Johns Hopkins University
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2010
Blood vessels function as conduits for the delivery of O2 and nutrients. Hypoxia-inducible factor 1 (HIF-1) mediates adaptive transcriptional responses to hypoxia/ischemia that include expression of angiogenic cytokines/growth factors by hypoxic cells and expression of cognate receptors for these ligands by vascular cells and their progenitors. Impairment of HIF-1-dependent responses to hypoxia is a major factor contributing to the impaired vascular responses to ischemia that are associated with aging and diabetes. © 2010 American Heart Association, Inc.
Working with human breast cancer cells and mice, scientists at The Johns Hopkins University say new experiments explain how certain cancer stem cells thrive in low oxygen conditions. Proliferation of such cells, which tend to resist chemotherapy and help tumors spread, are considered a major roadblock to successful cancer treatment. The new research, suggesting that low-oxygen conditions spur growth through the same chain of biochemical events in both embryonic stem cells and breast cancer stem cells, could offer a path through that roadblock, the investigators say. “There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells,” said Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center. “That gives us a few more possible targets for drugs that diminish their threat in human cancer.” A summary of the findings was published online March 21 in the Proceedings of the National Academy of Sciences. Semenza said scientists have long known that low oxygen environments affect tumor growth, but, in the case of advanced tumors, there was a paradox. “Aggressive cancers contain regions where the cancer cells are starved for oxygen and die off, yet patients with these tumors generally have the worst outcome. Our new findings tell us that low oxygen conditions actually encourage certain cancer stem cells to multiply through the same mechanism used by embryonic stem cells.” All stem cells are immature cells known for their ability to multiply indefinitely and give rise to progenitor cells that mature into specific cell types that populate the body’s tissues during embryonic development. They also replenish tissues throughout the life of an organism. But stem cells found in tumors use those same attributes and twist them to maintain and enhance the survival of cancers. According to Semenza, “Chemotherapy may kill more than 99 percent of the cancer cells in a tumor but fail to kill a small population of cancer stem cells that are responsible for subsequent cancer relapse and metastasis.” “The search has been intense to find these cells’ Achilles’ heel. If we could get cancer stem cells to abandon their stem cell state, they would no longer have the power to keep repopulating tumors,” said Semenza, who also directs the Vascular Biology Research Program at the Institute for Cell Engineering. Aiding their new research, Semenza said, was the knowledge that whereas the air we breathe is 21 percent oxygen, oxygen levels average around 9 percent in healthy human breast tissue but only 1.4 percent in breast tumors. Recent studies showed that low oxygen conditions increase levels of a family of proteins known as HIFs, or hypoxia-inducible factors, that turn on hundreds of genes, including one called NANOG that instructs cells to become stem cells. Studies of embryonic stem cells revealed that NANOG protein levels can be lowered by a chemical process known as methylation, which involves putting a methyl group chemical tag on a protein’s messenger RNA (mRNA) precursor. Semenza said methylation leads to the destruction of NANOG’s mRNA so that no protein is made, which in turn causes the embryonic stem cells to abandon their stem cell state and mature into different cell types. To see whether cancer stem cell renewal involves a chain of events similar to that used by embryonic stem cells, and whether the process was affected by oxygen levels, Semenza and graduate student Chuanzhao Zhang focused their studies on two human breast cancer cell lines that responded to low oxygen by ramping up production of the protein ALKBH5, which removes methyl groups from mRNAs. (Breast cancer is categorized and treated based on the presence or absence of three hormone receptors displayed on the outer membranes of cells. One human cell line they studied displays the receptors for estrogen and progesterone, and one, known as triple negative, displays none.) Zeroing in on NANOG, the scientists found that low oxygen conditions increased NANOG’s mRNA levels through the action of HIF proteins, which turned on the gene for ALKBH5, which decreased the methylation and subsequent destruction of NANOG’s mRNA. When they prevented the cells from making ALKBH5, NANOG levels and the number of cancer stem cells decreased. When the researchers manipulated the cell’s genetics to increase levels of ALKBH5 without exposing them to low oxygen, they found this also decreased methylation of NANOG mRNA and increased the numbers of breast cancer stem cells. Finally, using live mice, the scientists injected 1,000 triple-negative breast cancer cells into their mammary fat pads, where the mouse version of breast cancer forms. Unaltered cells created tumors in all seven mice injected with such cells, but when cells missing ALKBH5 were used, they caused tumors in only 43 percent (six out of 14) of mice. “That confirmed for us that ALKBH5 helps preserve cancer stem cells and their tumor-forming abilities,” Semenza said. Semenza said his team will continue its mouse studies to see if metastasis — the spread of cancer from the original tumor — is affected by the low oxygen/ALKBH5/NANOG relationship too. The researchers also want to see what other proteins and mRNAs are involved in the relationship, and why some cancer cell lines they tested did not show the same increased ALKBH5 levels in response to low oxygen levels.
Neuroscientists desperately need better tools to develop drugs for treating mental illness and neurodegenerative diseases. Experiments in lab animals more often than not produce drug candidates that ultimately fail in clinical trials. If a laboratory cell culture existed that accurately mimicked the intricate cellular networks that make up the cerebral cortex, it would provide a reality check and help answer the persistent question every drug developer wants to know: if this chemical works in mice that mimic some of the effects of schizophrenia or autism, will it work in humans? A team from Johns Hopkins Medical School and Nanjing University has just created a microcosm of the cerebral cortex that fits inside a lab dish. The “cortex in a dish” consists of an interwoven mesh of neurons that transmit electrical signals and other cells that damp down this activityThe cultured neurons may expand the toolkit needed to lift neurological and psychiatric drug development out of its present rut. Scientific American talked with Valina Dawson, co-director of the Institute for Cell Engineering at Johns Hopkins University School of Medicine, about a paper published April 6 in Science Translational Medicine on which she was the senior author. Scientific American: Describe what a 'cortex in a dish' is? Valina Dawson: The brain is divided into different structures and regions. The cerebral cortex is the largest part of the brain and manages higher brain functions such as thought and actions, language, sensory processing such as hearing and vision. One way to study how the neurons in the cortex function is to grow them in culture, “in a dish.” SA: Why have researchers wanted to create this laboratory model of the cortex? VD: Having human neurons in culture allows experimental investigations into signaling events at the chemical, protein and genetic levels that underlie normal and diseased actions in a manner that would not be possible or ethical in an intact human brain. Understanding how the neurons in the cortex work and what goes wrong when disease occurs will provide, we hope, new therapeutic opportunities to treat patients who suffer from brain injury and disease. SA: Why has it been so difficult to build this and how did you overcome hurdles along the way? VD: The ability to create human neurons from stem cells is relatively new. Established protocols to make different types of neurons from stem cells are limited to a few types of neurons out of the hundreds that exist in the human brain. Most of our understanding about the carefully controlled and elegantly choreographed events in the development of the cortex are from less complex organisms. Thus some of the key elements necessary to generate a protocol to produce the complex network of neuronal populations had to be guessed and identified by trial and error. SA:. What will scientists be able to do with your system? VD: We hope they will use this system to understand important mechanisms in cortical function and communication. We hope these cultures will also be useful in understanding how to provide neuroprotection against stroke and trauma and to investigate what goes wrong in diseases such as schizophrenia, autism and epilepsy. SA: Didn’t you show in the paper just published how this might work? Yes, in a model of stroke. Stroke is a common cause of death, disability and loss of quality of life world-wide but unfortunately there are few treatments to reduce the brain injury suffered. During a stroke there is a loss of blood flow to brain tissue. This can be mimicked in a culture dish by removing oxygen and glucose or by activating with the chemical NMDA a specific protein on the surface of neurons, an excitatory receptor. We have used our cortical culture system to study the cellular signaling events that occur and lead to neuronal cell death. Previously we found in rodent systems, that a member of a biochemical pathway, poly(ADP-Ribose) polymerase-1 (PARP-1) is pathologically activated and serves as a switch, directing the cell away from DNA repair towards cell death. Preventing PARP-1 activation protects neurons from ischemic cell death. For the first time we were able to determine using our cortical culture if human neurons respond in a similar manner, and they do! Inhibitors of PARP have been developed for the treatment of patients with certain types of cancer and some of these clinically useful drugs cross the blood brain barrier. Finding agents that can gain access to the brain has been a major hurdle in developing good treatments for neurologic disease and injury. Our studies raise the potential that these drugs could also be useful in the treatment of stroke. SA: What are the next steps? VD: Besides using these cultures to study how cortical neurons protect themselves from injury, we are also using these cultures to probe the molecular signaling events that underlie a form of autism with the hopes of finding a way to intervene. Additionally, other brain regions connect to the cortex, providing important information or receiving instructions from cortical regions. In the future, cultures could be established so that these connections could be studied at the cellular and sub-cellular level. One could also envision using sophisticated bioengineered scaffolds to permit the normal layering of the cortex so that interconnectivity between layers could be studied. In the science fiction future, perhaps cortical plugs would be developed that could be implanted into patients with stroke or trauma to replace the brain material that was damaged and lost.
News Article | April 23, 2016
The SpinΩ bioreactor used to create the conditions for mini-brains to grow.Image: John Hopkins Medicine Researchers from Johns Hopkins University have confirmed one of the most important ways that Zika causes microcephaly in the brains of infants via an innovative new technology, according to a study published Friday in Cell. By growing realistic brain organoids (groups of tissue meant to mimic actual organs) inside 3D printed bioreactors (vessels in which the brains are grown) the researchers confirmed that Zika infects specialized stem cells responsible for building the cerebral cortex, the outer layer of the brain. Zika then turns these cells into virus factories. The mini brains consisted of many different kinds of cells mixed together Xuyu Qian, the lead author of the paper and a PhD candidate at Johns Hopkins University told me. His team’s work showed that the virus prefers attacking neurological stem cells—the kind found in the cranium of developing fetuses—over other types. “In this paper we confirmed the preferential target of the virus,” Qian said. Their work also showed that the extent of the damage caused by Zika differs depending on when cells are infected. The "mini brains" survive for up to 100 days according to the study, allowing the researchers to examine what happens when a mother is infected very early and towards the middle of her pregnancy. Their work supported other clinical findings that suggest babies are most at risk during their first trimester, although there are still disastrous effects later in a mother’s pregnancy. A mini-brain infected with Zika virus. The virus is shown in green, vulnerable neural progenitor cells are shown in red, and neurons are shown in blue. The researchers grew the "brains" using human stem cells. A number of researchers in different fields have grown tiny organs for years, but the process is typically incredibly difficult due to the complexity of various organs and the high cost of the nutrients needed to cultivate human stem cells in the lab. In order to help overcome these hurdles, several high school students who spent a summer working in the Johns Hopkins University School of Medicine’s Institute for Cell Engineering came up with the idea for a smaller 3D printed bioreactor that would require less nutrients than the larger, commercially available options. After three years of testing different prototypes, the researchers were finally able to develop one that provided a suitable environment for the development of tiny brains. Dubbed SpinΩ, the paper’s authors included plans for it in their work, so that it could be recreated in other labs. “The design isn’t sophisticated, but it’s very intuitive,” Qian said. “This technology is not restricted to the Zika virus, it can be used to study all sorts of developmental disorders,” Qian told me. In the future, he hopes to commercialize the bioreactor that his team created in order to lower its price. “We hope it becomes a standard model for studying human development,” he said.