In recent years, Dr. Harold Brem’s laboratory has conducted and published seminal research into the mechanisms of action and effectiveness of cellular therapies developed to promote diabetic foot ulcer healing. Our specific accomplishments include the following:
MicroRNAs are small molecules of ribonucleic acid that regulate gene expression in the cell nucleus. Dr. Brem’s laboratory identified a set of microRNAs that are expressed abnormally in chronic venous ulcers, and demonstrated that these molecules are involved in cell processes important to foot ulcer healing. This finding has resulted in testing of potential therapeutic agents that target these molecules. We believe that these sequences may represent potential therapeutic targets to improve healing of diabetic foot ulcers. We have developed small molecules that can target these microRNAs in the cell, and are seeking funding to test these molecules to determine whether they are effective in overcoming wound repair defects. We have filed for a patent on these molecules, and have an NIH grant application for a study testing these molecules that has been scored and is awaiting approval pending the submission of additional data.
Using animal models, researchers in Dr. Brem’s lab demonstrated that when diabetic ulcers are treated topically with a sustained-release formulation of granulocyte macrophage colony stimulating factor (GM-CSF), the treatment resulted not only in increased ketartinocyte migration, fibroblast proliferation and collagen deposition, but also promoted proper alignment of collagen—making it more stable and less susceptible to degradation. This growth factor is one of several regenerative therapies that we now use regularly as part of our treatment protocols.
We discovered that vascular endothelial growth factor (VEGF), a powerful new drug originally developed to help blood vessels grow, also stimulates skin cell regeneration in chronic wounds—a finding that has influenced the use of growth factors in treatment and has also supported further clinical investigation in diabetes.One of the most potent growth factors for stimulating angiogenesis, VEGF has already been approved by the FDA for use in treating patients with angina. We used mouse models of diabetes to demonstrate that local delivery of VEGF directly to an open wound accelerates the healing rate and restores normal skin function. We then tested VEGF on human cells cultured from the wound tissue of patients with diabetic foot ulcers and reported that it accelerates epithelialization, granulation tissue formation, and collagen production.
VEGF appears to work in multiple ways: by promoting migration of specialized skin cells into the wound bed, where they grow and fill in the open wound, and also by stimulating development of normal, well-ordered skin structures that surround the cells and maintain the skin’s function and resilience. In some of these studies, VEGF was delivered to the open wound via gene therapy in order to maintain active VEGF at the site long enough for the growth factor to have optimal effect.
“Human skin equivalent” (also called “cellular therapy”) is a skin construct containing layers of the two major types of skin cells suspended within a collagen matrix that is widely used to promote closure in long-standing open skin wounds. When sheets of this material are placed over open chronic skin wounds of patients with diabetic foot ulcers, they stimulate faster and more complete healing.
In addition to reporting on the efficacy of this treatment for such patients, our studies both in the laboratory and with patients have demonstrated that human skin equivalent, previously thought to act as a type of inert skin graft, in fact is biologically active and provides 15 growth factors that are important for skin repair. These growth factors are released by the living cells and act together in a synchronized manner to accelerate healing through the controlled release of growth factors from keratinocytes and fibroblasts, which attract, stimulate and support the growth of the specialized skin cells needed to reconstitute the complex structure of normal skin. This therapy decreases amputations in persons with diabetic foot ulcers and is the only FDA-approved treatment for venous ulcers that is based on randomized controlled clinical trials. The discovery of its underlying mechanisms has had a major impact in terms of encouraging clinical research into cellular therapies for chronic wounds.
Some time ago, our researchers made the unexpected discovery that synthesis of glucocorticoid (GC) hormone is increased in chronic wounds, specifically in the non-healing edge of such wounds, where it contributes to inflammation and impaired healing. This finding has already led us to patent a novel drug therapy for chronic wounds, utilizing topical glucocorticoid antagonists.
Further investigation revealed that this pathway involves glucocorticoid hormone-mediated degradation of GSK3b, nuclear translocation of β-catenin, and activation of c-myc. Within minutes, through the mediation of the enzymes PKC and PLC, it phosphorylates the enzyme GSK3b, subsequently leading to its degradation. We have shown 1) that there is increased activity of GC and decreased amounts of GSK3b in the epidermis of patients with chronic wounds, and 2) that downstream targets of this pathway, β-catenin, and c-myc, are activated in the epidermis of patients with chronic wounds. This means that GC-mediated phosphorylation of GSK3b plays an important role in the pathogenesis of a chronic ulcer, and that protecting GSK3b from phosphorylation and degradation—for example, through the topical use of PKC and PLC inhibitors (such as Calphostine C or U073122) or RU486 (GR antagonist)—would promote wound healing in patients with chronic ulcers. When we tested this novel therapeutic approach by applying a PKC inhibitor topically on an induced wound, it promoted healing. Untreated wounds healed 50% on day 4 post-wounding, whereas treated wounds completely healed. Taken together, the combination of RU486 and PKC inhibitors may prevent activation of the β-catenin–c-myc pathway. Furthermore, this approach can potentially be combined with pro-angiogenic therapy, stimulating epithelialization and restoring impaired angiogenesis in order to promote healing of chronic wounds.
The longer that cellular therapy or growth factors can remain active after being applied topically, the more effective they will be and the less often patients will need undergo additional surgical procedures to reapply them. Our lab has demonstrated that delivery of the gene for vascular endothelial growth factor (VEGF) in an adenovirus (ADV) vector to skin wounds in mouse models of diabetes resulted in accelerated wound healing. Since then, we have also used this method to deliver the growth factor GM-CSF to wounds. In addition, our research team has developed a novel line of biodegradable polymers that release regenerative biological agents into wound tissue over a sustained period of time as they disintegrate. These polymers have also been used successfully to deliver VEGF and GM-CSF) to wounds in animal models..