Anti-Diabetes Drug Acts With Stem Cells to Repair Heart After Heart Attack


Exenatide is the generic name of a antidiabetes drug whose market name is Byetta. Made by Eli Lilly and Company, Exenatide binds to a receptor on the surface of insulin-secreting pancreatic beta cells and stimulates the insulin response of those cells. Insulin is the main hormone that tells the cells in our bodies to take up sugar and use it. Type 2 diabetics, however, have cells that have become de-sensitized to insulin and they require more insulin to signal to the cells to metabolize sugar, Once a type 2 diabetic injects himself with Exenatide, the body secretes more insulin than it might normally secrete, Thus, type 2 diabetics are able to more effectively control their blood sugar levels with this drug.

A) The molecular structure of human GLP-1. B) The molecular structure of exenatide (gray colors indicate differences in structure from human GLP-1. C) The molecular structure of liraglutide (gray colors indicate changes in structure from human GLP-1).
A) The molecular structure of human GLP-1. B) The molecular structure of exenatide (gray colors indicate differences in structure from human GLP-1. C) The molecular structure of liraglutide (gray colors indicate changes in structure from human GLP-1).

Normally, these receptors are bound by a small protein that is made by cells in the small intestine called GLP-1 (glucagon-like peptide-1). The small intestine makes GLP-1 when it is exposed to sugar, and it is the combination of high levels of sugar in the blood, plus the presence of GLP-1 that causes the pancreatic beta cells to release insulin. In type 2 diabetics, the beta cells release insulin, but the body is de-sensitized to it. Therefore, more insulin is needed to control the blood sugar levels. Exenatide does just that by acting like GLP-1.

What does this have to do with stem cells and regenerative medicine? It turns out that the heart also has GLP-1 receptors on the surfaces of its cells and the binding of GLP-1 to these receptors decreases inflammation in the heart, prevents the death of heart muscle cells, protects blood vessels, and protects against damage from reactive oxygen species (also known as free radicals). A fascinating paper has appeared in the Journal of Cellular and Molecular Medicine from the Chinese PLA Hospital in Beijing, China.  In this publication, Chinese cardiologists used a compound that is closely related to Exenatide called Exendin-4 in combination with stem cells to determine if the activation of the GLP-1 receptor influences the healing qualities of stem cells after a heart attack.

In this paper, Yundai Chen and colleagues extracted stem cells from the fat tissue of rats.  This fat tissue was minced and then the stem cells were isolated on the basis of cell surface proteins that are common only to stem cells in fat.  Then Chen and his co-workers gave heart attacks to 120 rats and divided the rats into five groups, with 30 animals each.  The first group received injections of buffer into their hearts immediately after the induction of a heart attack.  The second group received injections of Extendin-4 three days prior to the heart attack and seven days after.  The third group received injections of fat-derived stem cells into the heart tissue bordering the infarcted tissue.  The fourth group received the stem cell injections plus the treatments with Extendin-4.

Exendin-4 is one of the compounds extracted from the salivary glands of the Gila monster, a colorful lizard (shown below)  found in the deserts of California.  Exenatide is the acetate salt of Extendin-4, and both compounds bind the GLP-1 receptor and elicits a biological response.

Gila Monster

 

The results of these experiments were as follows:  the animals that received buffer injections showed respectable amounts of cell death and oxidative damage in their hearts.  However, those animals that received Extendin-4 injections showed less oxidative damage and significantly less cell death.  The same could be said for those animals that received the fat-derived stem cell treatments.  However, those animals that were treated with Extendin-4 plus the stem cells showed substantially less oxidative damage and cell death than all the other groups.

The heart function tests show similar trends.  The ejection fraction, which measures the percentage of the blood that comes into that heart that is pumped out, was in the cellar in the buffer-injected animals, about 10% higher in the Extendin-4 and stem cell-treated animals, and almost 20% higher in the animals treated with both Extendin-4 and the stem cells.   The degree to which the heart muscle contracted (reported as % of shortening or fractional shortening) was over double that in the dually treated animals.  Also the size of the heart scar in the dually treated animals was half the size observed in the animals treated with buffer.

Further examinations of the heart of the dually treated animals showed that the fat-derived stem cells expressed genes normally found in blood vessels cells and heart muscle cells.  This is not definitive evidence that these cells differentiated into heart-specific cell types, but they clearly are surviving and doing something beneficial to the heart.

In culture, the fat-derived stem cells made a whole host of healing molecules when they are treated with Extendin-4.  Also, Extendin-4 treatment protected with stem cells from being damaged by noxious chemicals (e.g., hydrogen peroxide).  Biochemical studies showed that the stem cells that had been treated with Extendin-4 had activated the STAT3 pathway.  Why is this significant?  Because the STAT3 is normally activated by cells when they are stressed.  It is a “I want to survive” kind of pathway.  Extendin-4 seems to cause the stem cells to kick into high gear, survive better, and heal better.

A scheme illustrating the potential cardioprotective signalling pathways through which exenatide may reduce myocardial infarct size and protect the heart against lethal myocardial reperfusion injury. The actual mechanism underlying the cardioprotective effects elicited by exenatide remains to be elucidated, although it is assumed that many of the beneficial effects are mediated through the activation of the glucagon-like peptide-1 (GLP-1) receptor on the cardiomyocytes. The activation of this receptor then recruits pro-survival signalling cascades such as the phosphatidylinositol 3-kinase (PI3K)–Akt and adenylate cyclase (AC)–cAMP–protein kinase A (PKA) pathways which protect the heart against acute ischaemia–reperfusion injury through a number of potential mechanisms including: the inhibition of the mitochondrial permeability transition pore (mPTP), the activation of AKAPs (protein kinase A-anchoring proteins), increased myocardial glucose uptake (possibly via p38 mitogen-activated protein kinase and iNOS), reduced apoptotic cell death, and the transcription of cardioprotective factors (such as PPAR-β/δ, Nrf-2, and HO-1). eNOS, endothelial nitric oxide synthase; GSK, glycogen synthase kinase; HO-1, haem oxygenase 1; iNOS, inducible nitric oxide synthase; NO, nitric oxide; Nrf-2, nuclear respiratory factor 2; PKC, protein kinase; PKG, protein kinase G; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species.
A scheme illustrating the potential cardioprotective signalling pathways through which exenatide may reduce myocardial infarct size and protect the heart against lethal myocardial reperfusion injury. The actual mechanism underlying the cardioprotective effects elicited by exenatide remains to be elucidated, although it is assumed that many of the beneficial effects are mediated through the activation of the glucagon-like peptide-1 (GLP-1) receptor on the cardiomyocytes. The activation of this receptor then recruits pro-survival signalling cascades such as the phosphatidylinositol 3-kinase (PI3K)–Akt and adenylate cyclase (AC)–cAMP–protein kinase A (PKA) pathways which protect the heart against acute ischaemia–reperfusion injury through a number of potential mechanisms including: the inhibition of the mitochondrial permeability transition pore (mPTP), the activation of AKAPs (protein kinase A-anchoring proteins), increased myocardial glucose uptake (possibly via p38 mitogen-activated protein kinase and iNOS), reduced apoptotic cell death, and the transcription of cardioprotective factors (such as PPAR-β/δ, Nrf-2, and HO-1). eNOS, endothelial nitric oxide synthase; GSK, glycogen synthase kinase; HO-1, haem oxygenase 1; iNOS, inducible nitric oxide synthase; NO, nitric oxide; Nrf-2, nuclear respiratory factor 2; PKC, protein kinase; PKG, protein kinase G; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species.

Now, these experiments were performed in rodents.  Therefore, it remains to be seen if the FDA-approved Exenatide will improve stem cell survival as well as the unapproved Extendin-4.  While they should have the same biological activity, different preparations of the similar molecules can elicit different responses.  Also, it is unclear if this strategy would work in humans.  Do human fat-derived or even bone marrow-derived stem cells respond in a similar fashion to Extendin-4 or Exenatide?  Would such an experiment work in humans?  Do the risks associated with Exenatide administration outweigh the potential benefits of administering it?  Could the stem cells simply be pre-treated with Exenatide before being administered?

This paper has truly opened up a can of worms that should keep scientists busy for many years to come.

Primed Fat-Based Stem Cells Enhance Heart Muscle Proliferation


A Dutch group from the University of Groningen has shown that fat-based stem cells can enhance the proliferation of cultured heart muscle cells. The stem cells used in these experiments were preconditioned and this pretreatment greatly enhanced their ability to activate heart muscle cells.

This paper, by Ewa Przybyt, Guido Krenning, Marja Brinker, and Martin Harmsen was published in the Journal of Translational Medicine. To begin, Przybyt and others extracted human adipose derived stromal cells (ADSC) from fat tissue extracted from human liposuction surgeries. To do this, they digested the fat with enzymes, centrifuged and washed it, and then grew the remaining cells in culture.

Then they used rat neonatal heart muscle cells and infected them with viruses that causes them to glow when certain types of light was shined on them. Then Przybyt and others co-cultured these rat heart cells with human ADSCs.

In the first experiment, the ADSCs were treated with drugs to prevent them from dividing and then they were cultured with rat heart cells in a one-to-one ratio. The heart muscle cells grew faster with the ADSCs than they did without them. To determine if cell-cell contact was required for this stimulation, they used the culture medium from ADSCs and grew the heart cell on this culture medium. Once again, the heart cells grew faster with the ADSC culture medium than without it. These results suggest that the ADSCs stimulate heart cell proliferation by secreting factors that activate heart cell division.

Another experiment subjected the cultured heart cells to the types of conditions they might experience inside the heart after a heart attack. For example, heart cells were subjected to low oxygen tensions (2% oxygen), and inflammation – two conditions found within the heart after a heart attack. These treatments slowed heart cell growth, but this heart cell growth was restored by adding the growth medium of ADSCs. Even more remarkably, when ADSCs were grown in low-oxygen conditions or treated with inflammatory molecules (tumor necrosis factor-alpha or interleukin-1beta), the culture medium increased the fractions of cells that grew. Therefore, ADSCs secrete molecules that increase heart muscle cell proliferation, and increase proliferation even more after the ADSCs are preconditioned by either low oxygen tensions or inflammation.

In the next experiment, Przybyt and others examined the molecules secreted by ADSCs under normal or low-oxygen tensions to ascertain what secreted molecules stimulated heart cell growth. It was clear that the production of a small protein called interleukin-6 was greatly upregulated.

Could interleukin-6 account for the increased proliferation of heart cells? Another experiment showed that the answer was yes. Cultured heart cells treated with interleukin-6 showed increased proliferation, and when antibodies against interleukin-6 were used to prevent interleukin-6 from binding to the heart cells, these antibodies abrogated the effects of interleukin-6.

Przybyt and others then took these results one step further. Since the signaling pathways used by interleukin-6 are well-known, they examined these pathways. Now interleukin-6 signals through pathways, once of which enhances cell survival, and another pathway that stimulated cell proliferation. The cell proliferation pathway uses a protein called “STAT3” and the survival function uses a protein called “Akt.” Both pathways were activated by interleukin-6. Also, the culture medium of ADSCs that were treated with interleukin-6 induced the interleukin-6 receptor proteins (gp80 and gp130) in cultured heart muscle cells. This gives heart muscle cells a greater capacity to respond secreted interleukin-6.

This paper shows that stromal stem cells from fat has the capacity, in culture, to activate the growth of cultured heart muscle cells. Also, if these cells were preconditioned with low oxygen tensions or pro-inflammatory molecules, those fat-based stem cells secreted interleukin-6, which enhanced heart muscle cell survival, and proliferation, even if those heart muscle cells are exposed to low-oxygen tensions or inflammatory molecules.

This suggests that preconditioned stem cells from fat might be able to protect heart muscle cells and augment heart healing after a heart attack. Alternatively, cardiac administration of interleukin-6 after a heart attack might prove even more effective to protect heart muscle cells and stimulate heart muscle cell proliferation. Human trials anyone?