Thymosin beta4-Overexpressing Cells Heal Heart After a Heart Attack


Thymosin beta4 is a very highly conserved 43-amino acid peptide that plays a very important role in cell proliferation, migration, and angiogenesis (blood vessel production). Experiments with thymosin beta4 in laboratory animals that have had a heart attack have shown that treatment with thymosin beta4 can reduce cell death in the heart and reduce the size of the infarct, while increasing heart function (see Hannappel E, et al., Arch Biochem Biophys 240 (1985): 236-241; Bock-Marquette, et al., Nature 432 (2007): 466-472; Srivastava D, et al., Ann NY Acad Sci 1112 (2007): 161-170; Grant DS et al., Angiogenesis 3 (1999): 125-135). Also, knocking down thymosin beta4 in endothelial progenitor cells (cells that make blood vessels) prevents these cells from healing the heart after a heart attack (Hinkel, et al., Circulation 117 (2008): 2232-2240).

Thymosin beta4
Thymosin beta4

Given the ability of thymosin beta4 to heal the heart, Dinender Singla and colleagues at the University of Central Florida have engineered embryonic stem cells to express thymosin beta4 and used them to treat laboratory animals that have suffered a heart attack. The results were truly tremendous.

Singla and his team genetically engineered mouse embryonic stem cells to express either red fluorescent protein or red fluorescent protein and thymosin beta4. In culture, those cells that expressed thymosin beta4 showed much more efficient differentiation into heart muscle cells (3-5 times greater).

Effect of Tβ4 Expression on ES Cell Differentiation. A. Fluorescent microscopy of EBs derived from RFP-ES and Tβ4-ES cells. At D12 EBs were stained with anti- sarcomeric α-actin (S-actin) (green) and counterstained with DAPI for nuclear visualization (blue). The lower panel shows S-actin staining in a beating area (square box) in the EBs derived from Tβ4-ES cells. Scale = 200µm. B. Percentage of beating EBs during cardiac myocyte differentiation. Spontaneously beating EBs were examined and counted under a light microscope at D9, 12 and 15. C. Real-time PCR analysis of gene expression of GATA-4, Mef2c and Tbx6 at D12. Data are represented as mean ± SEM, *p< 0.05; vs. RFP ESCs.
Effect of Tβ4 Expression on ES Cell Differentiation.
A. Fluorescent microscopy of EBs derived from RFP-ES and Tβ4-ES cells. At D12 EBs were stained with anti- sarcomeric α-actin (S-actin) (green) and counterstained with DAPI for nuclear visualization (blue). The lower panel shows S-actin staining in a beating area (square box) in the EBs derived from Tβ4-ES cells. Scale = 200µm. B. Percentage of beating EBs during cardiac myocyte differentiation. Spontaneously beating EBs were examined and counted under a light microscope at D9, 12 and 15. C. Real-time PCR analysis of gene expression of GATA-4, Mef2c and Tbx6 at D12. Data are represented as mean ± SEM, *p< 0.05; vs. RFP ESCs.

Next, they gave laboratory mice heart attacks and implanted these cells into the heart. Those mice that received no cells had bucket loads of cell death. Those mice who received embryonic stem cells that did not express thymosin beta4 showed a decrease in cell death 2 weeks after the heart attack. However those mice that received the embryonic stem cells that expressed thymosin beta4 showed a third of the cell death found in the control mice. The same applied to the amount of scarring in the hearts. Animals treated with embryonic stem cells (ESCs) that did not express thymosin beta4 had about half the scarring of the control mice that received no cells, but the hearts treated with thymosin beta4-expressing ESCs showed about a third of the scarring.

Transplanted Tβ4-ES Cells Reduce Cardiac Fibrosis in the Infarcted Mouse Heart. A. Representative photomicrographs of tissue sections stained with Masson’s trichrome at D14 post MI surgery. Scale =100µm. B. Quantitative analysis of interstitial fibrosis for control and experimental groups. #p<0.05 vs. sham, *p<0.05 vs. MI, and $p<0.05 vs. RFP-ESCs. C. Histogram illustrates quantitative MMP-9 expression. #p<0.05 vs sham, *p<0.05 vs. MI. n = 5-7 animals per group.
Transplanted Tβ4-ES Cells Reduce Cardiac Fibrosis in the Infarcted Mouse Heart.
A. Representative photomicrographs of tissue sections stained with Masson’s trichrome at D14 post MI surgery. Scale =100µm. B. Quantitative analysis of interstitial fibrosis for control and experimental groups. #p

When it came to heart function, things were really remarkable. The ESC-treated hearts showed definite improvement over the control animals, but the ESC-thymosin beta4 cells restored heart function so that the hearts worked almost as well as the sham hearts that were never given a heart attack. The fractional shortening was not as high, nor was the end diastolic volume as low, but most of the other functional parameters were close to the sham hearts.

Transplanted Tβ4-ES Cells Improve Cardiac Function in the Infarcted Heart. Echocardiography was performed D14 following MI. A. Raw functional data. Histograms show average quantified measurements of B. left ventricular internal diameter during diastole (LVIDd) C. left ventricular internal diameter during systole (LVIDs) D. fractional shortening FS% E. end diastolic volume (EDV) F. end systolic volume (ESV) G. and ejection fraction EF% at 2 weeks after MI for all treatment groups. #p<0.05 vs. sham, *p<0.05 vs. MI, and $p<0.05 vs. RFP-ESCs. Data set are from n=6-8 animals/group.
Transplanted Tβ4-ES Cells Improve Cardiac Function in the Infarcted Heart.
Echocardiography was performed D14 following MI. A. Raw functional data. Histograms show average quantified measurements of B. left ventricular internal diameter during diastole (LVIDd) C. left ventricular internal diameter during systole (LVIDs) D. fractional shortening FS% E. end diastolic volume (EDV) F. end systolic volume (ESV) G. and ejection fraction EF% at 2 weeks after MI for all treatment groups. #p

Mechanistically, the thymosin beta4 appears to down-regulate PTEN and upregulated the AKT kinase. AKT kinase activation is associated with cell survival and growth. PTEN tends to slow down growth and prevent healing under some conditions.

Effects of Tβ4 Expression on Caspase-3, pAkt, and p-PTEN Activities. Heart homogenates from each group were prepared for ELISA analysis of caspase-3, Akt, and p-PTEN. A. Quantitative analysis of caspase-3, B. p-PTEN, and C. pAkt activity in the hearts following cell transplantation. Data were represented as Mean ± SEM; *p<0.01 vs. MI, #p<0.05 vs. sham. n = 4-5 animals per group.
Effects of Tβ4 Expression on Caspase-3, pAkt, and p-PTEN Activities.
Heart homogenates from each group were prepared for ELISA analysis of caspase-3, Akt, and p-PTEN. A. Quantitative analysis of caspase-3, B. p-PTEN, and C. pAkt activity in the hearts following cell transplantation. Data were represented as Mean ± SEM; *p

This suggests that thymosin beta4 expression seems to augment healing in the heart after a heart attack. Such a therapy could potentially be used to treat heart attack patients, however, more animal experiments will need to be done. What is the proper time frame for thymosin beta4 treatment? How many cells should be implanted in order to provide the maximum therapeutic effect. Can such a treatment be provided via intracoronary delivery? Can conditional expression provide a robust enough response to heal the heart? Can other cells, like mesenchymal stem cells to used to deliver the thymosin beta4? Can c-kit cardiac progenitor cells be used to deliver thymosin beta4?

Many questions remain, but hopefully, this remarkable treatment regime can be ramped up to eventually go to clinical trials.

Gene Inhibitor Plus Fish Fibrin Restore Nerve Function Lost After a Spinal Cord Injury


Scientists at UC Irvine’s Reeve-Irvine Research Center have discovered that injections of salmon fibrin injections into the injured spinal cord plus injections of a gene inhibitor into the brain restored voluntary motor function impaired by spinal cord injury.

Gail Lewandowski and Oswald Steward, director of the Reeve-Irvine Research Center at UCI, examined rodents that had received spinal cord injuries.  They were able to heal the damage by developmentally turning back the clock in a molecular pathway that is critical to the formation of the corticospinal nerve tract, and by providing a scaffold for the growing neurons so that the axons of these growing neurons could grow and make the necessary connections with other cells.  Their research was published in the July 23 issue of The Journal of Neuroscience.

The work of Steward and Lewandowski is an extension of previous research at UC Irvine from 2010.  Steward and his colleagues discovered that the axon of neurons grow quite well once an enzyme called PTEN is removed from the cells.  PTEN is short for “phosphatase and tensin homolog,” and it removes phosphate groups from specific proteins and lipids.  In doing so, PTEN signals to cells to stop dividing and it can also direct cells to undergo programmed cell death (a kind of self-destruct program).  PTEN also prevents damaged tissues from regenerating sometimes, because it is a protein that puts the brakes of cell division.  Mutations in PTEN are common in certain cancers, but the down-regulation of PTEN is required for severed axons to re-form, extend, migrate to their original site, and form new connections with their target cells.

PTEN function

 

After two years, team from U.C. Irvine discovered that injections of salmon fibrin into the damaged spinal cord or rats filled cavities at the injury site and provided the axons with a scaffolding upon which they could grow, reconnect and facilitate recovery. Fibrin produced by the blood system when the blood vessels are breached and it is a fibrous, insoluble protein produced by the blood clotting process.  Surgeons even use it as a kind of surgical glue.

“This is a major next step in our effort to identify treatments that restore functional losses suffered by those with spinal cord injury,” said Steward, professor of anatomy & neurobiology and director of the Reeve-Irvine Research Center. “Paralysis and loss of function from spinal cord injury has been considered irreversible, but our discovery points the way toward a potential therapy to induce regeneration of nerve connections.”

In their study, Steward and Lewandowski subjected rats to spinal cord injuries, and then assessed their defects.  Because these were upper back injuries, the rats all showed impaired forelimb (hand) movement.  Steward and Lewandowski then treated these animals with a combination of salmon fibrin at the site of injury and a modified virus that made a molecule that inhibited PTEN.  These viruses were genetically engineered adenovirus-associated viruses encoded a small RNA that inhibited translation of the PTEN gene (AAVshPTEN).  This greatly decreased the levels of PTEN protein in the neurons.  Other rodents received control treatments of only AAVshPTEN and no salmon fibrin.

The results were remarkable.  Those rats that received the PTEN inhibitor alone showed no improvement in their forelimb function, but those animals who were given AAVshPTEN plus the salmon fibrin recovered forelimb use (at least reaching and grasping).

“The data suggest that the combination of PTEN deletion and salmon fibrin injection into the lesion can significantly enhance motor skills by enabling regenerative growth of corticospinal tract axons,” Steward said.

Corticospinal Nerve tract

Statistics compiled by the Christopher & Dana Reeve Foundation suggests that approximately 2 percent of Americans have some form of paralysis that is the result of a spinal cord injury.  Spinal cord injuries break connections between nerves and muscles or nerves and other nerves.  Even injuries the size of a grape can cause complete loss of function below the level of the injury.  Injuries to the neck can cause paralysis of the arms and legs, an absence of sensation below the shoulders, bladder and bowel incontinence, sexual dysfunction, and secondary health risks such as susceptibility to urinary tract infections, pressure sores and blood clots due to an inability to move one’s legs.

Steward said the next objective is to learn how long after injury this combination treatment can be effectively administered. “It would be a huge step if it could be delivered in the chronic period weeks and months after an injury, but we need to determine this before we can engage in clinical trials,” he said.

Adult Stem Cells Suppress Cancerous Growth While Dormant


William Lowry and his postdoctoral fellow Andrew White at UCLA’s Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research have discovered the means by which particular adult stem cells suppress their ability to trigger skin cancer during their dormant phase. A better understanding of this mechanism could provide the foundation to better cancer-prevention strategies.

This study was published online Dec. 15 in the journal Nature Cell Biology. William Lowry, Ph.D. is an associate professor of molecular, cell and developmental biology in the UCLA College of Letters and Science.

Hair follicle stem cells are those tissue-specific adult stem cells that generate the hair follicles. Unfortunately, they also are the cell population from which cutaneous squamous cell carcinoma, a common skin cancer, begins. However, these stem cells cycle between active periods, when they grow, and dormant periods, when they do not grow.

Diagram of the hair follicle and cell lineages supplied by epidermal stem cells. A compartment of multipotent stem cells is located in the bulge, which lies in the outer root sheath (ORS) just below the sebaceous gland. Contiguous with the basal layer of the epidermis, the ORS forms the external sheath of the hair follicle. The interior or the inner root sheath (IRS) forms the channel for the hair; as the hair shaft nears the skin surface, the IRS degenerates, liberating its attachments to the hair. The hair shaft and IRS are derived from the matrix, the transiently amplifying cells of the hair follicle. The matrix surrounds the dermal papilla, a cluster of specialized mesenchymal cells in the hair bulb. The multipotent stem cells found in the bulge are thought to contribute to the lineages of the hair follicle, sebaceous gland, and the epidermis (see red dashed lines). Transiently amplifying progeny of bulge stem cells in each of these regions differentiates as shown (see green dashed lines).
Diagram of the hair follicle and cell lineages supplied by epidermal stem cells. A compartment of multipotent stem cells is located in the bulge, which lies in the outer root sheath (ORS) just below the sebaceous gland. Contiguous with the basal layer of the epidermis, the ORS forms the external sheath of the hair follicle. The interior or the inner root sheath (IRS) forms the channel for the hair; as the hair shaft nears the skin surface, the IRS degenerates, liberating its attachments to the hair. The hair shaft and IRS are derived from the matrix, the transiently amplifying cells of the hair follicle. The matrix surrounds the dermal papilla, a cluster of specialized mesenchymal cells in the hair bulb. The multipotent stem cells found in the bulge are thought to contribute to the lineages of the hair follicle, sebaceous gland, and the epidermis (see red dashed lines). Transiently amplifying progeny of bulge stem cells in each of these regions differentiates as shown (see green dashed lines).

White and Lowry used transgenic mouse models for their work, and they inserted cancer-causing genes into these mice that were only expressed in their hair follicle stem cells. During the dormant phase, the hair follicle stem cells were not able to initiate skin cancer, but once they transitioned into their active period, they began growing cancer.

Dr. White explained it this way: “We found that this tumor suppression via adult stem cell quiescence was mediated by PTEN (phosphatase and tensin homolog), a gene important in regulating the cell’s response to signaling pathways. Therefore, stem cell quiescence is a novel form of tumor suppression in hair follicle stem cells, and PTEN must be present for the suppression to work.”

Retinoids are used to treat certain types of leukemias because they drive the cancer cells to differentiate and cease dividing. Likewise, understanding cancer suppression by inducing quiescence could, potentially, better inform preventative strategies for certain patients who are at higher risk for cancers. For example, organ transplant recipients are particularly susceptible to squamous cell carcinoma, as are those patients who are taking the drug vemurafenib (Zelboraf) for melanoma (another type of skin cancer). This study also might reveal parallels between squamous cell carcinoma and other cancers in which stem cells have a quiescent phase.

Breast Cancer Clinical Trial Targets Cancer Stem Cells


Even though my previous posts about cancer stem cells have generated very little interest, understanding cancer as a stem cell-based disease has profound implications for how we treat cancer. If the vast majority of the cells in a tumor are slow-growing and not dangerous but only a small minority of the cells are rapidly growing and providing the growth the most of the tumor, then treatments that shave off large numbers of cells might shrink the tumor, but not solve the problem, because the cancer stem cells that are supplying the tumor are still there. However, if the treatment attacks the cancer stem cells specifically, then the tumor’s cell supply is cut off and the tumor will wither and die.

In the case of breast cancer, the tumors return after treatment and spread to other parts of the body because radiation and current chemotherapy treatments do not kill the cancer stem cells.

This premise constitutes the foundation of a clinical trial operating from the University of Michigan Comprehensive Cancer Center and two other sites. This clinical trial will examine a drug that specifically attacks breast cancer stem cells. The drug, reparixin, will be used in combination with standard chemotherapy.

Dr. Anne Schott, an associate professor of internal medicine at the University of Michigan and principal investigator of this clinical trial, said: “This is one of only a few trials testing stem cell directed therapies in combination with chemotherapy in breast cancer. Combining chemotherapy in breast cancer has the potential to lengthen remission for women with advanced breast cancer.”

Cancer stem cells are the small number of cells in a tumor that fuel its growth and are responsible for metastasis of the tumor. This phase 1b study will test reparixin, which is given orally, with a drug called paclitaxel in women who have HER2-negative metastatic breast cancer. This study is primarily designed to test how well patients tolerate this particular drug combination. However, researchers will also examine how well reparixin appears to affect various cancer stem cells indicators and signs of inflammation. The study will also examine how well this drug combination controls the cancer and affects patient survival.

This clinical trial emerged from laboratory work at the University of Michigan that showed that breast cancer stem cells expressed a receptor on their cell surfaces called CXCR1. CXCR1 triggers the growth of cancer stem cells in response to inflammation and tissue damage. Adding reparixin to cultured cancer stem cells killed them and reparixin works by blocking CXCR1.

Mice treated with reparixin or the combination of reparixin and paclitaxel had significantly fewer (dramatically actually) cancer stem cells that those treated with paclitaxel alone. Also, riparixin-treated mice developed significantly fewer metastases that mice treated with chemotherapy alone (see Ginestier C,, et al., J Clin Invest. 2010, 120(2):485-97).