Testosterone Replacement Treatment Improves Circulation in the Hearts of Rats that Have Suffered a Heart Attack

Prior to going through menopause, women have fewer heart attacks and other types of heart-related events than men. This is almost certainly due to the protective effects of estrogen on the cardiovascular system (Kolovou G, et al., Curr Vasc Pharmacol. 2011;9(2):244-57). However, once women experience a heart attack, they actually fare more poorly than men (Vaccarino V, et al., Arch Intern Med 2009;169:1767-74 and Daly C, et al., Circulation 2006;113:490-8). This reason for this seems to due to the male sex hormone testosterone.

Several studies provide evidence of the pro-blood vessel-making effects of the hormone testosterone. First of all, several large clinical studies have shown that men with low testosterone levels have an increased risk of cardiovascular diseases and mortality (Shores MM, et al., Arch Intern Med 2006;116(15):1660-5;Khaw KT, et al., Circulation 2007;116:2694-2701; Laughlin GA, et al., J. Clin Endocrinol Metab 2008;93:68-75). Secondly, a meta-analysis of these data has also supported the role of testosterone in supporting male cardiovascular health (Kintzel PE, et al., Pharmacotherapy 2008;28:1511-22). Finally, males show higher levels of collateral circulation in their hearts after a heart attack than women who have suffered a heart attack, which also supports a role for testosterone in the induction of new blood vessels formation in the heart (Abaci A, et al., Circulation 1999;99:2239-42).

Given these data, a Chinese research group has used a rat model to examine the ability of testosterone to induce new blood vessels growth after a heart attack. Yeping Chen and Lu Fu and their co-workers from the Harbin Medical University in Harbin, China have published a paper in the European Journal of Pharmacology that addressed this issue. Their results are fascinating and might bring new implications for post-heart attack cardiac therapy in men.

In the experiment, Chen and Fu and colleagues used rats as a model system. They took 100 male laboratory rats and broke them into two groups. Rats in the first group were surgically castrated, and rats in the second group underwent the castration surgery but without actual castration (known as a sham castration procedure). Then the rats were broken into four groups. The first group was sham castrated rats, the second was castrated rats that had been given placebos, the third was castrated rats that received testosterone supplementation (2 mg / kg body weight), and the fourth group consisted of castrated rats that received testosterone and an anti-testosterone drug called flutamide. All drug treatments commenced on the same day as the castration procedure.

One to three days after the castration procedure, peripheral blood samples were taken from all rats to measure the levels of CD34+ stem cells. CD34+ stem cells make blood vessels, and the levels of these stem cells in circulating blood are an indication of how well these animals make new blood vessels.

All rats were given two weeks to recover from the castration surgery and then were given heart attacks. Four weeks later, all rats were subjected to electrocardiograms and then some were sacrificed and their heart tissues were examined microscopically.

The results of these experiments were quite telling. Groups 1 and 3 rats (the sham castrated rats and castrated rats that had received testosterone supplementation) and group 4 rats (castrated rats that had received testosterone and flutamide) had significantly more circulating CD34+stem cells than group 2 rats (castrated rats that received no testosterone supplementation). Testosterone raised the level of circulating CD34+ stem cells and flutamide did not reverse this effect. Flutamide (Eulexin, Flutamin) works by competing with testosterone and its active metabolite dihydrotestosterone for the androgen receptor in prostate gland cells. For this reason, flutamide is categorized as an “anti-androgen” drug. It is given orally for prostate cancer in males and is also used to treat polycystic ovary syndrome in females, since it can reduce androgen levels in women.

The increase in CD34+ stem cells was due to testosterone-induced increases in the expression of several pro-blood vessel-inducing molecules. These molecules that induce new blood vessels are called “angiogenic factors.” The angiogenic factors induced by testosterone in group 1, 3, and 4 rats, but not in group 2 rats, include HIF1a (hypoxia-induced factor-1a), SDF-1 (stromal cell derived factor-1), and VEGF (vascular endothelial growth factor). The expression of these angiogenic factors is significantly increased in group 1, 3, & 4 rats, but not group 2. Furthermore, tissue examinations of hearts from all four groups show that hearts from groups 1, 3, & 4 have significantly greater quantities of CD34+ stem cells infiltrating them than those from group 2.

Echocardiograms of hearts from rats from all four groups show that groups 1, 3, and 4 had significantly smaller areas of cell death than hearts from rats in group 2. Cell death assays showed similar results as well.

Functional aspects of the heart also showed that hearts from rats in groups 1, 3, & 4 functioned more efficiently than hearts from rats from group 3.

These results suggest that testosterone could improve the blood vessel production in the heart of males after a heart attack. These data also suggest that this mechanism by which testosterone does this is independent of the androgen receptor found in the prostate gland. Therefore, if patients have a family risk of prostate cancer, a drug like flutamide can be given with the testosterone to improve the circulation in the damaged heart without increasing the risk of the patient for prostate cancer. Perhaps a clinical trial should be proposed to examine the effects of testosterone in human heart attack patients.

Smart Bomb-Type Drug Successfully Treats Advanced Breast Cancer in Clinical Trials

In a key clinical trial with 1,000 women who had advanced breast cancer, the efficacy of an experimental treatment for breast cancer has been examined in some detail. This breast cancer treatment is one of the first “smart bomb” treatments for breast cancer.

This treatment uses a drug to deliver a toxic payload to tumor cells that also leaves the healthy cells alone. In this treatment, woman with advanced disease this experimental “smart bomb” treatment extended the lives of these sick women by several months, and during this time, the women lived without their cancer getting worse. After two years, 65 percent of women who received this treatment were still alive versus 47 percent of those in a comparison group who were given two standard cancer drugs. The developers of this treatment plan to report on it at a cancer conference in Chicago.

“The absolute difference is greater than one year in how long these people live,” said the study’s leader, Dr. Kimberly Blackwell of Duke University. “This is a major step forward.”

How does this treatment work? It builds on the cancer drug, Herceptin. Herceptin was developed as a gene-targeted therapy for breast cancer. In fact, it was the first gene-targeted treatment ever developed. Herceptin is used for about 20 percent of patients whose tumors overproduce a certain protein. Herceptin is the trade name for the drug trastuzumab, and this drug is a monoclonal antibody. Trastuzumab binds to a protein called “human epidermal growth factor receptor 2” or Her2. Her2 has several other names (Neu, ErbB2, CD340 or p185), but whatever you call it, Her2 binds a small protein called epidermal growth factor (EGF). The binding of EGF to Her2 sets a series of events into action inside the cell that causes it to grow and divide vigorously.

Her2 is overexpressed in breast cancer cells and the excessive signaling to the cell interior drives the breast cancer cells to grow faster and faster. Trastuzumab binds to Her2 and shuts the signaling that emanates from it down. Trastazumab is used as part of a treatment regimen that includes drugs like Adriamycin® (doxorubicin), Cytoxan® (cyclophosphamide), and either Taxol® (paclitaxel) or Taxotere® (docetaxel). Such a treatment course is known as “AC→TH.” Other treatment regimens include Herceptin with Taxotere and Paraplatin® (carboplatin). This treatment course is known as “TCH.”

In this “smart bomb” treatment, researchers linked trastuzumab to a toxin that kills cells once it gets inside them. This new drug is called T-DM1, the “smart bomb.” Herceptin binds the toxin to the cancer cells, and the toxin provides the coup de gras for the cancer cell. Doctors tested T-DM1 in 991 women who suffered from breast cancer that had spread throughout their body and was getting worse despite treatments with available chemotherapy and ordinary Herceptin. The women in this study were given T-DM1 infusions every three weeks or infusions of standard breast cancer treatment drugs (Xeloda plus daily Tykerb pills). The median time until cancer worsened was nearly 10 months in the women given T-DM1, in comparison to just over 6 months for those who received the other treatments. According to Blackwell, this is about the same magnitude of benefit initially seen with Herceptin, which later proved to improve overall survival
Even more interestingly, T-DM1 caused fewer side effects than the other drugs. Unfortunately, some women on T-DM1 showed had signs of liver damage and poor blood clotting, but most patients did not show the usual problems of chemotherapy. According the Blackwell, “People don’t lose their hair, they don’t throw up. They don’t need nausea medicines, they don’t need transfusions.”

Dr. Michael Link, a pediatric cancer specialist at Stanford University who is president of the American Society of Clinical Oncology, the group hosting the Chicago conference where the results were being presented, said “The data are pretty compelling. It’s sort of a smart bomb kind of therapy, a poison delivered to the tumor … and not a lot of other collateral damage to other organs.”

Dr. Louis Weiner, director of Georgetown Lombardi Comprehensive Cancer Center, said the results strongly suggest T-DM1 improves survival, since it delivers more drug directly to tumors with less side effects. This is a clear advance over other chemotherapeutic drugs.

Denise Davis, 51, a customer service representative at a propane company, was diagnosed three years ago with breast cancer that had spread to her liver and bones. Since February of last year, the Lynchburg, Va., woman has made the two-hour trip to Duke in Durham, N.C., every three weeks to get infusions of T-DM1. Davis called T-DM1 “Herceptin-plus.” Cancer scans every six weeks show her tumors are either shrinking or stable. Davis concluded, “Right now, I’m feeling pretty good about it. The only way I’d feel a little better is if it took care of everything, but I’ll take what I can get.”

Genentech, part of the Swiss company Roche, plans to seek approval later this year to sell the drug in Europe and the United States. Another company, ImmunoGen Inc., made the technology that combined the two drugs. Genentech says the price of T-DM1 has not been determined. Herceptin costs more than $4,000 a month plus whatever doctors charge to infuse it. Herceptin’s U.S. patent will not expire until 2019.

Platelet-Lysate Bioactive Scafold for Tissue Engineered Cartilage

Cartilage replacement at joints represents a tremendous challenge for regenerative medicine. While growing cartilage in culture is possible, scaling this technology up to generate enough high-quality articular cartilage (the kind of cartilage found at joints), is still a distinct challenge. To date, stem cell treatments can heal small breaches in cartilage, but reconstructing large lesions is still not possible. In general, cartilage at joints has very poor healing properties, and therefore, is a major challenge in orthopedics.

A major improvement in therapeutics is the use of a technique called “autologous chondrocyte implantation” or ACI. ACI involves the delivery of healthy cartilage-making cells (chondrocytes) from the patient’s own body after these cells have been grown and expanded in culture. In order to coax these cartilage-making cells to make cartilage, special scaffolds are used that provide a three-dimensional matrix upon which the chrondrocytes grow and form cartilage. These 3-D scaffolds are essential to keep the chondrocytes differentiated and making cartilage.

One of the most promising types of scaffolds for making cartilage are “bioactive 3D scaffolds.” These types of scaffolds can deliver growth factors and other molecules to the chrondrocytes and boost their growth and cartilage production.

In a recent publication, Andrei Moroz and colleagues in the Extracellular Matrix Laboratory at the Botucatu Institute of Biosciences, São Paulo State University, Brazil, have used mesenchymal stem cells (MSCs) from rabbit bone marrow and differentiated them into chondrocytes. This allowed them to use stem cells from bone marrow instead of harvesting cartilage from the joints, which can be very painful and deleterious to the joint. The main innovation in this paper was the use of a platelet-lysate-based 3D bioactive scaffold to support the chondrogenic differentiation and maintenance of MSCs.

MSCs from adult rabbit bone marrow were isolated, characterized, and grown in 60 microliters of platelet lysate from rabbit blood. Platelets are very small cells from circulating blood that assist in the formation of clots that staunch bleeding after a blood vessel in damaged. Platelets are easy to isolate from circulating blood and the rabbit platelet-lysate clot scaffold was maintained is a standard tissue culture medium (Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12) that was supplemented with other molecules known to induce cartilage formation in MSCs. After three weeks in culture, the MSCs were examined in detail. Not only were they nice and round, but they were filled with cartilage-specific molecules, and clumped together like chondrocytes.

According to this research group, they are on to something with this platelet-lysate bioactive scaffold. It provided a suitable system for culturing MSCs and allowed them to make lots of cartilage. The scaffold also was easy to make, and maintained the MSCs in a cartilage-making state without causing cell death or stressing the cells. Therefore, it might provide an alternative to autologous chondrocyte implantation. The next steps in this research will be to use this engineered cartilage to repair damaged joints to see if the cartilage made by cells embedded in platelet-lysate 3D bioactive scaffolds can act as functional cartilage.

For the article see Andrei Moroz, et al., Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering.  Platelets. 2012.

Fat-Derived Mesenchymal Stem Cells Engineered to Express Heme Oxidase-1 Improve Heart Function After a Heart Attack in Rabbits

Fat-derived mesenchymal stem cells (MSCs) are easily procured and have the ability to differentiate into a variety of tissue, including heart muscle. When implanted into the hearts of laboratory animals, fat-derived MSCs induce the formation of new heart muscle and blood vessels and improve the function of the heart (Hwangbo, et al., Yonsei Med J 2010;51:69-76; Lin et al., J Transl Med 2010;8:88 & Yu, et al., Int J Cardiol 2010;139:166-172). Unfortunately, once implanted into the heart, the majority of cells undergo programmed cell death. The inhospitable environment of the heart after a heart attack is simply to hostile to support the growth and differentiation of these cells.

Are there ways to help the implanted cells and prevent them from dying before they have had a change to help the heart? The answer to this question is an unequivocal “yes.” MSCs can be genetically engineered to express a variety of molecules that help them resist hostile conditions (see Conrad P. Hodgkinson, et al., “Genetic Engineering of Mesenchymal Stem Cells and Its Application in Human Disease Therapy,” Hum Gene Ther. 2010; 21(11): 1513–1526). In one paper, Jun-jie Yang and colleagues in the laboratory of Yun-dai Chen in the Department of Cardiology at the Chinese PLA Hospital General Hospital engineered fat-derived MSCs with a gene called “heme oxygenase-1 (HO-1) to help the cells resist hostile conditions. They then used these engineered cells to treat heart attacks in rabbits.

The HO-1 gene encodes an enzyme that participates in dis-assembly of heme. Hemoglobin carries oxygen in the blood from the lungs to the tissues, and hemoglobin consists of a protein backbone known as the “globin” part of hemoglobin, and a flat, planar, molecule that holds an iron atom at its center known as “heme.” Hemoglobin is not the only protein that contains heme, since several other iron-utilizing proteins also possess heme groups for binding iron. When iron-utilizing binding proteins are degraded, the iron is removed and recycled, the protein portion is degraded and the amino acids that compose it are also recycled. Heme, however, is not recycled. Free heme is damaging to the cell, and heme is degraded in two reactions to bilirubin. The enzymes that degrade heme are heme oxygenase (which converts heme to biliverdin) and biliverdin reductase (which converts biliverdin to bilirubin. In our bodies, bilirubin is conjugated to acidic sugar, which increases its solubility and allows excretion.

Cells with high levels of HO-1 are able to endure higher levels of stress and adverse conditions. As it turns out, HO-1 degrades many other things besides heme, and this seems to be the reason why HO-1 confers on cells a greater ability to withstand stressful conditions (see Shibahara, Tohoku J Exp Med 2003;200:167-86 & Zeng B., et al., J Biomed Sci 2010;17:80).

In this paper, three groups of nine rabbits were given a echocardiogram, which measures the activity of the heart muscle and then two of the groups were given liposuction, and then rabbits in all three groups were given heart attacks. The fat-derived MSCs were isolated and cultured, and the MSCs from the first group were transduced with HO-1. Then 13 days after the induction of the heart attack, all the rabbits were given a second echocardiogram and then 14 days after the heart attack, the rabbits were given either MSCs transduced with HO-1, MSCs, or buffer with no cells. All MSCs were directly injected into the heart muscle. Then 42 days after the induction of the heart attack, all 27 rabbits were given a final echocardiogram and then sacrificed for tissue examinations of the hearts..

The results showed that the Ho-1 engineered MSCs were much less likely to die and also showed a greater ability to resist hydrogen peroxide, an agent that is known to kill cells. When the hearts from the three groups were examined, it was clear that before the cell treatments, there were no functional differences between the hearts in the animals of either group. However, 42 days after induction of the heart attack, and one month after cell treatment, the hearts of the HO-1/MSC group had smaller areas of dead cells, great blood vessel density, greater connectivity between the heart muscle cells, and better innervation by the sympathetic nervous system.

Even though both groups that received MSCs showed better functioning hearts than those in the control group that received only buffer injections,l the HO-1/MSC group had hearts that were functionally superior to those in the MSC only group. Tissue examinations of the hearts of animals from the HO-1/MSC group also showed staining in the injected areas for proteins associated with blood vessels and heart muscle. This strongly suggests that the implanted fat-derived MSCs are differentiating into heart muscle and blood vessels.

These data show that even though MSC implantations can improve the function of hearts from laboratory animals that have suffered heart attacks, engineered MSCs can improve the heart even more and are safe and efficacious. Hopefully, this will spur the FDA to approve human clinical trials that use engineered MSCs for heart attack patients.

Amgen’s New Drug Blinatumomab Shows Success in ALL Patients

Amgen Corporation announced updated results from its Phase 2 study with blinatumomab. Blinatumomab is a specially produced antibody that targets a protein called “CD19.” This antibody is made by an engineered cell line that produces one and only one kind of antibody. Such an antibody is called a “monoclonal antibody.” Monoclonal antibodies are made by antibody-making cells (B-lymphocytes) that are fused to tumor cells. The tumor cell immortalizes the B lymphocyte and this immortal cell now makes one type of antibody for its entire existence. Such a cell line that results from the fusion of a tumor cell with a B lymphocyte is called a “hybridoma” cell line.

CD19 is a cell surface protein that is made on the surfaces of B lymphocytes. Because B lymphocytes can over-grow and form blood-based tumors, an antibody that binds tightly to CD19 can specifically target B lymphocyte-based tumors. The binding of such antibodies also alerts other immune cells (T cells) to home to those cells and destroy them.

Blinatumomab, however, is an even more special molecule, because it binds CD19 at one end of the protein and a T cell-specific protein called CD3. Blinatumomab, therefore, acts as a bridge between tumor cells and T cells. It helps the T cells recognize the tumors as foreign. It is therefore an unusual type of chemotherapeutic agent called a bi-specific T-cell engager or BiTE. Another BiTE is MT110:, which is used to treat gastrointestinal and lung cancers, and is directed against the EpCAM antigen and the T cell surface protein B3.

Treatment with blinatumomab helped achieve a high-rate of complete response (CR) in 72% of all adult patients who were diagnosed with relapsed or refractory B-precursor acute lymphoblastic leukemia (ALL), and were treated in the study.

Full results of the study will be presented at the 48th Annual Meeting of the American Society of Clinical Oncology (ASCO) on June 4, 2012.

For more information on this Phase 2 single-arm dose-ranging clinical trial, 26 of the 36 patients treated with blinatumomab (across all of tested doses and schedules) achieved a complete response with partial recovery of their blood cell counts. All but two patients achieved a “molecular response..” Molecular response means that the presence of leukemic cells were not detectable with polymerase chain reaction (PCR) assays. There were also not treatment-related deaths or serious adverse events reported in this study.

Median survival was 9.0 (8.2, 15.8) months with a median follow-up period of 10.7 months at the time of the analysis. In the group of patients who received the selected dose of blinatumomab, the median survival time was 8.5 months, and the median duration of response in the 26 patients who responded to treatment was 8.9 months.

Max Topp, department of internal medicine II, University of Wuerzburg and chair of the study, said: “For these patients with limited treatment options, the remission rate observed in the trial is a vast improvement over the current standard of care. These results also represent significant progress in our research of immunotherapies; a new approach to fighting cancer that we believe could make a real difference for patients.”

Patients who received the selected dose and schedule, the most common adverse events were mild and included fever, (70%), headache (39%), shaking (30%) and fatigue (30%). Reversible central nervous system events led to treatment interruptions in six patients with two patients permanently discontinuing treatment.

Cardiophere-Derived Cells Embedded in Platelet Gel Increases Heart Function and Improves Heart Structure After a Heart Attack

Biomaterials are organic compounds that can be molded into the shape of a particular organ or tissue, and can be seeded with cells that will form the shape of the organ or tissue and degrade the it, while using the biomaterial as a scaffold for their growth and development.

One organ where biomaterials can make a great difference is the heart, since implanted cells tend to either die to move away from the heart. By implanting cells into the heart that are embedded in biomaterials, the implanted cells stay put, are protected from cell death induced by the inhospitable environment of the heart after a heart attack, and tend to differentiate into heart-specific cells at the site at which they were implanted.

Injectable biomaterials are preferable for the heart, since non-injectable biomaterials require that the surgeon crack the chest and implant the biomaterial, which is a much more invasive procedure. One of the most appealing injectable biomaterials is platelet gel (also known as platelet fibrin scaffold).

The body naturally generates platelet gel after injury, however, it can be engineered as a tissue substitute to speed healing. The scaffold for platelet gel consists of naturally occurring biomaterials composed of a cross-linked fibrin network.

Platelet gel polymerization requires the enzyme thrombin and its substrate fibrinogen. Thrombin degrades fibrinogen to fibrin, which self-assembles to form the fibrin meshwork that composes the ground substance for platelet gel.  This reaction is affected primarily by the concentration of thrombin and temperature. Platelet gels are composed of fibers whose thicknesses vary according to the reaction conditions, and can be enriched by addition of other molecules (fibronectin, vitronectin, laminin, and collagen). Linking these molecules to the fibrin scaffold greatly affects the properties of the platelet gel, and the gel can also serve as a reservoir for growth factors and other molecules that speed healing.

Injection of platelet gel into a heart that has just experienced a heart attack prevents remodeling. Can stem cells that have a documented ability to heal damaged hearts have their healing capacities increased by implanting them in platelet gel?

A paper published workers in Eduardo Marban’s lab at Cedars-Sinai Medical Center in Los Angeles in the journal Biomaterials asks this very question, using rats as a model. In this article, Marban’s group used cardiosphere-derived cells (CDCs), which were successfully used in the CADUCEUS clinical trial to heal the hearts of human patients who have suffered a heart attack. The strategy used in these experiments was relatively simple (in principle): Induce heart attacks in the rats, treat once group with platelet gel alone, and the other group with CDCs embedded in platelet gel. Then compare the structural and functional integrity of the hearts in each group.

The results of these experiments come in several categories. First of all, the CDCs grown in platelet gel showed increased viability (reduced death) in comparison to CDCs grown on standard tissue culture plates. Furthermore, platelet gel-grown CDCs also differentiated into three-dimensional structures such as blood vessels. The CDCs also degraded the platelet gel and by two weeks of culture, two-thirds of the platelet gel was degraded. Furthermore, CDCs in platelet gel spread out and began to beat. Far more CDCs spread out and beat when grown in platelet gel than those grown in tissue culture plates. The contraction of the CDC-formed heart muscle cells was also much more robust in platelet gel than in tissue culture plates. Overall, the CDCs did much better in platelet gel than in standard tissue culture plates. They grew better, survived better, formed more heart-specific structures, and differentiated in more mature heart cell types when grown in platelet gel.

Another bonus to the platelet gel consists in its ability to trap growth factors. The CDCs in the platelet gel secrete a wide variety of growth factors, and these growth factors bind to the platelet gel and are concentrated by it. This recruits other cells to the platelet gel. That increases the ability of the platelet gel to facilitate stem cell-mediated healing.

Implanting platelet gel alone and platelet gel seeded with CDCs into damaged hearts caused increased heart wall thickness, decreased infarct size, and improved cardiac function. However in all cases, the CDC-seeded platelet gel causes even greater improvements than platelet gel alone.

These experiments show that stem cell-mediated healing is improved by the use of biomaterials. Furthermore, platelet gel is a very easily manufactured biomaterial that improves the growth and heart-specific differentiation of CDCs. Give the demonstrated healing capacities of CDCs, augmenting those capabilities with biomaterials such as platelet gel should be a priority for future clinical trials.