MicroRNAs hold promise for treating diseases in blood vessels

A newly discovered mechanism controls whether muscle cells in blood vessels hasten the development of both atherosclerosis and Alzheimer's disease, according to an article published online today in the journal Nature.

The study was led by the Gladstone Institute of Cardiovascular Disease (GICD) in San Francisco, with key contributions from the Aab Cardiovascular Research Institute at the University of Rochester School of Medicine and Dentistry.

Thanks to stem cells, humans develop from a single cell embryo into a complex being with about 250 unique cell types. As the fetus develops, cells divide and multiply (proliferate) in many generations and specialize (differentiate) with each generation until millions of functional cells result (bone, nerve, blood, skin, muscle, etc.). To serve specific roles in the body, some stem cells also switch back and forth between primitive, rapidly proliferating precursors and their mature, functioning, non-proliferating counterparts, a quality called "plasticity."

Among the most "plastic" of cells are vascular smooth muscle cells (VSMC), which form in layers around blood vessels, and by contracting or relaxing, regulate blood pressure. Because VSMC surround blood vessels that are continually becoming clogged by atherosclerosis, they must be ever ready to grow along with the vessel as it attempts, by growing, to remain open to blood flow despite fatty deposits and inflammation. If these efforts fail, heart attack or stoke may occur. Each time a vessel grows to avoid a clog, the VSMC surrounding it must grow too by reverting to their high-growth precursor form. Once a vessel reaches its growth limit, however, the growth that once kept vessels open begins adding to clogs by thickening vessel walls.

Past studies in Rochester have shown that transition of VSMC from fast-proliferating stem cells to mature cells and back is largely controlled by two proteins, myocardin and serum response factor (SRF), as part of a regulatory network that influences many genes. SRF anchors to certain snippets of DNA, while myocardin turns on the genes to which SRF sticks. Most of the genes turned on by myocardin/SRF in VSMC are needed for normal function. When levels of myocardin decrease, as they do for some reason in vascular diseases like atherosclerosis, VSMC no longer work normally and vessel thickening ensues. For this reason the field has sought urgently to learn how myocardin levels are controlled, but without success.

Enter a research team led by Deepak Srivastava, M.D, director of GICD, world leaders in the characterization of microRNAs (miRNAs). These small, single-stranded molecules of ribonucleic acid (RNA), discovered in the Victor Ambros lab in 1993, fine-tune protein levels in all cells of the body. The GICD team discovered that miRNAs control VSMC differentiation and growth.

Gene expression is the process where information encoded in genes is converted into proteins, the workhorse molecules that make up the body's structures and carry its signals. While genes are encoded in chains of deoxyribonucleic acids (DNA), they are copied into chains of messenger ribonucleic acids (mRNA) that are "read" by cellular machines that build proteins. microRNAs bind to messenger RNAs, usually targeting them for breakdown or rendering them unfit to serve as templates for protein production.

The current study found that two miRNAs in particular, miR-143 and miR-145, are part of a molecular switch that determines whether VSMC persist as high-growth precursors or mature into functioning muscle cells. miR-143 was found to block the expression of factors that promote proliferation by VSMC precursors. Surprisingly, miR-145 activated the expression of myocardin, which maintains VSMC in their mature form over their high-growth form.

In a mouse model, expression of miR-143 and miR-145 was reduced to almost nothing where disease-related proliferation of VSMC had thickened blood vessel walls. These findings suggest that miR-143 and miR-145 – in partnership with myocardin – maintain the normal balance between mature VSMC and their precursors. Thus, researchers believe the drop in miR-143 and miR-145 levels seen in disease settings contributes greatly to vessel wall thickening, but that theory will need to be confirmed by further studies.

In addition, Rochester investigators found that myocardin and SRF activate genes that may influence the rate at which the brain can remove amyloid beta, the toxic protein that builds up in blood vessels in the brains of patients with Alzheimer's disease. In a February 2009 article in Nature Cell Biology, University of Rochester investigator, Berislav Zlokovic, M.D., Ph.D. found that when SRF and myocardin are active, amyloid beta accumulates in VSMC lining blood vessels. The discovery that miR-145 encourages the expression of myocardin could explain why myocardin may occur in higher levels in Alzheimer's disease, which is turning out to be a problem of "vascular plumbing."

"The finding that a microRNA controls levels of myocardin, the master regulator of VSMC identity and function, forms the starting point in efforts to design new classes of treatment for vascular diseases that represent leading causes of death," said Joseph M. Miano, Ph.D., associate professor within the Aab Cardiovascular Research Institute at the University of Rochester Medical Center, and a study author. He and Srivastava trained together under the direction of renowned muscle biologist Eric Olson at M.D. Anderson Cancer Center in the early 1990s. Miano was also a co-author of the paper on Alzheimer's with Zlokovic. "One of the most important of potential applications for this work would be to deliver miR-145 into vessel walls as a way to normalize levels of myocardin, which would counter vessel wall thickening."

Rochester provided GICD with samples of blood vessels containing lesions with dramatically reduced levels of myocardin. GICD then looked at levels of miR-143 and miR-145 in this disease setting. The team in Rochester also did experiments to show that local delivery of miR-145 in mouse blood vessels leads to elevated expression of myocardin and its target genes.

Little it takes to tip the balance of p53

A tightly controlled system of checks and balances ensures that a powerful tumor suppressor called p53 keeps a tight lid on unchecked cell growth but doesn't wreak havoc in healthy cells. In their latest study, scientists at the Salk Institute for Biological Studies suggest just how finely tuned the system is and how little it takes to tip the balance.

When unprovoked, at least two negative regulators—the related proteins Mdm2 and Mdmx—prevent p53 from unleashing its power to kill. But just slightly increasing the amount of available Mdmx, which grips p53 and renders it inactive, the Salk researchers discovered, made mice remarkably resistant to the harmful effects of radiation but very susceptible to the development of oncogene-induced lymphomas.

"Our experiments emphasize how subtle and precarious the balance is," says postdoctoral researcher and first author Yunyuan V. Wang. "A slight shift of balance and the mice survive the equivalent of Chernobyl but are in big trouble when an oncogene is activated."

Their findings, to be published in the July issue of the journal Cancer Cell, could explain why some tumors don't respond to radiation or chemotherapy, and provide novel routes for the development of new anti-cancer therapies.

As a powerful tumor suppressor, p53 turns on genes that either halt cell division to allow time for repair of damaged DNA or, when all rescue attempts prove futile, to prevent cells with genetic defects from dividing, as this would fuel the development of cancer. Consequently, before any tumor cell can start proliferating willfully, it needs to escape from p53's iron fist.

"One way or another, p53 function is compromised in all cancers. Either p53 itself is mutated or there is a problem with one of the proteins that regulate p53's activity," says the study's leader Geoffrey M. Wahl, Ph.D., a professor in the Gene Expression Laboratory. "Our hope is that we can develop small molecule drugs that will activate p53 in those tumors where it is still functional but inactivated by one of its negative regulators."

In an earlier study, Wahl and his team discovered that Mdm2 and Mdmx cooperate to prevent p53 from being activated, with Mdm2 being primarily responsible for degrading p53, while Mdmx is more effective at preventing p53 from turning on genes. But how p53 shakes off its negative regulators when cells experience one of the myriad stresses that activate p53 has been the topic of much discussion.

One view holds that after DNA damage occurs, enzymes directly modify p53 and that those modifications change the structure of p53 in such a way that neither Mdm2 nor Mdmx are able to bind. In an alternative scenario the same enzymes—kinases that attach phosphate groups to proteins—modify the negative regulators, accelerating their degradation and freeing p53 of their antagonists. Of course, it may well be that both views are correct, but the extent to which each contributes to p53 control remains an important unanswered question.

To get to the bottom of the dispute, Wang genetically engineered mice to eliminate three key phosphorylation sites in Mdmx. "The mutations stabilize Mdmx and as a result we saw consistently lower basal activity of p53. Surprisingly, we observed no increase in spontaneous tumor formation " she says. "In the absence of catastrophic DNA damage these low levels of p53 were enough to suppress tumorigenesis."

But in order to put cells on notice or commandeer them to commit suicide in the face of irreparable damage, these animals need to activate p53, which in turn activates a whole range of target genes. "Levels of active p53 still go up," says Wang, "but they never reach the critical threshold that's required to elicit a biological response."

As a result, these animals became very resistant to the deleterious effects of high doses of radiation. When blasted with 10 Gy of irradiation—enough to wipe out all blood stem cells in the bone marrow of normal mice—mutant mice that were unable to fully activate p53 experienced only a modest blood count drop. The other noticeable effect was a premature graying of their coat.

"Both radiation and chemotherapy are commonly used for the treatment of cancer and act by inducing DNA damage and subsequent cell death through p53. As such, tumors that retain normal p53 are more likely to respond to treatment while tumors carrying a defective p53 pathway are often less responsive ," says Wahl. Ideally, we want to find a therapeutic target, such as MDM2 or MDMX, that would increase p53 activity in tumor cells while minimally impacting other vital functions such as hematopoiesis."

Since p53 also protects against wayward cell proliferation caused by oncogenes such as c-myc, the researchers permanently activated c-myc in the B cell lineage to mimic human endemic Burkitt's lymphomas. They observed that mice with defective Mdmx developed very aggressive lymphomas at a very young age. Thus, control by Mdmx is critical to balance the severity of the response to DNA damaging agents, while also preventing induction of cancer by activated oncogenes.

New targeted therapy finds and eliminates deadly leukemia stem cells

New research describes a molecular tool that shows great promise as a therapeutic for human acute myeloid leukemia (AML), a notoriously treatment-resistant blood cancer. The study, published in Cell Stem Cell, describes exciting preclinical studies in which a new therapeutic approach selectively attacks human cancer cells grown in the lab and in animal models of leukemia.

AML is a cancer of the white blood cells that has an extremely poor prognosis and does not respond well to conventional chemotherapy. "The cellular and molecular basis for this dismal picture is unclear," offers senior study author Associate Professor Richard Lock from the Children's Cancer Institute Australia and the University of New South Wales. "However, previous research has suggested that leukemia stem cells (LSCs) may lie at the heart of post-treatment relapse and chemoresistance." LSCs are cells that can initiate AML and are critical for its long-term growth.

Associate Professor Lock and colleagues exploited the fact that the molecule CD123 is expressed at very high levels on LSCs but not on normal blood cells. CD123 is part of the interleukin-3 receptor, a protein that interacts with a growth factor (called a cytokine) that influences cell survival and proliferation. The researchers created a therapeutic antibody that recognized and bound to CD123 with the hope that this antibody would selectively interfere with AML-LSC survival.

When AML-LSCs from human patients were transplanted into mice treated with the antibody, called 7G3, cytokine signaling in the tumor cells was blocked. Further, 7G3 impaired migration of the AML-LSCs to bone marrow and activated the innate immune system of the host mouse to destroy the AML-LSCs. Overall, treatment with 7G3 substantially improved mouse survival when compared with control groups. The researchers go on to report that a CD123-targeting antibody is currently being used in phase 1 clinical trials of advanced AML and that there are no signs of treatment-related toxicity.

These results hold substantial promise for future cancer therapeutics. "The recent characterization of defined populations of cancer stem cells in a range of human malignancies, as well as their relative resistance to conventional chemotherapy and radiotherapy, supports the broad applicability of our approach and provides rationale for the progression of AML-LSC-targeted therapeutics from preclinical evaluation to clinical trials," concludes Associate Professor Lock.

New clue into how brain stem cells develop into cells which repair damaged tissue

Multiple sclerosis is an autoimmune disease which is caused by the body's immune system attacking nerve fibres and their protective insulation, the myelin sheath, in the central nervous system. This damage prevents the nerves from 'firing' properly, and then leads to their destruction, resulting in physical and intellectual disabilities.

It is currently thought that two components determine clinical outcomes in MS. First, it is important to stop ongoing damage (mainly achieved by controlling inflammation in the central nervous system). The second is to repair the damage that has occurred to the protective myelin sheaths surrounding the nerve fibres (this involves a regenerative process called remyelination in which new myelin sheaths are restored to nerve fibres).

While there exist several effective treatments to reduce inflammatory damage, no treatments are available to augment remyelination to repair the damage to nerve fibres. Critical to the development of such repair therapies is to understand how the brain's own stem cells can replace the myelin forming cells (oligodendrocytes) lost in the disease. During early stages of the disease the brains own stem cells are surprisingly good at repairing damage in MS. However, for reasons that until now have not been well explained, they become less efficient as the disease progresses.

In this study the researchers have identified the Wnt pathway, which plays an active role in the maintenance and proliferation of stem cells, as a crucial determinant of whether oligodendrocytes can efficiently make myelin. Their studies demonstrate that if the Wnt pathway is abnormally active, then the process is inhibited. This opens up the exciting possibility that the repair can be enhanced in MS patients by drugs that block the Wnt pathway.

Professor Robin Franklin from the University of Cambridge, a co-senior author of the study, explained the significance of their findings: "The pathway we identified plays a critical role in whether repair to the damaged cells will or will not occur. Interestingly, mutations in this particular pathway are also involved in several cancers. In this regard, drugs that inhibit this pathway from signaling have been sought which might suppress tumour growth. These same drugs may also find a role in promoting repair in MS."

Lead author of the study, Stephen Fancy, PhD, a postdoctoral fellow in the lab of co-senior author David Rowitch, MD, PhD, a Howard Hughes Medical Institute Investigator at the University of California, San Francisco, said: "We believe we have made a significant step forward in understanding why repair might fail in neurological diseases such as MS by identifying a pathway which inhibits the myelin repair process," said the

MS Society Director of Research, Jayne Spink, said: "We are delighted with the outcome of this outstanding research, which gives us greater knowledge of the mechanics of MS. This works opens up new avenues of research and lends itself to more study. Being able to uncover the secrets behind the damage caused in MS will take us forward in our understanding of this debilitating condition."

"Our studies work have implications for other diseases," said UCSF's Rowitch. "In a condition called periventricular leukomalacia (PVL), which can lead to cerebral palsy in extremely premature infants, recent studies show a similar inability of oligodendrocytes to perform their important repair function. In respect to failed myelin repair, we see a parallel between the chronic demyelinated plaques of multiple sclerosis and the lesions of PVL."