According to the World Health Organization, an estimated 422 million people worldwide suffer from diabetes, which was responsible for 1.5 million patient deaths in 2015 alone.1 Diabetic foot ulcers (DFUs) are a common and often debilitating complication of diabetes. In the United States, estimates suggest that between two and six million people will develop a diabetic foot ulcer annually.1 This is a major health concern as the World Health Organization estimates that the risk of lower extremity amputation is higher in diabetic patients with foot ulcers in comparison to non-diabetic patients.2
Patients with diabetes will often develop peripheral neuropathy. This loss of sensation can lead to the development of pressure ulcers on their feet. The standard of care, including sharp debridement, offloading and wound dressings, will only heal about fifty percent of diabetic foot ulcers at best with advanced therapy only healing about 40 percent of the remaining failed cases.3,4 Accordingly, there is a need for new therapies that better target the pathophysiology of diabetic wound healing.
What You Should Know About The Pathophysiology Of Diabetic Wound Healing
When a healthy non-diabetic patient develops an acute wound, whether it is through trauma or surgery, it heals through a highly regulated process of cell replication, migration, protein synthesis, matrix deposition and remodeling. This involves the synchronization of numerous growth factors, cytokines and various cell types, including keratinocytes, fibroblasts, myofibroblasts and macrophages.5-8
In chronic diabetic foot ulcers, this pathway fails or becomes stalled, and these wounds can remain dormant for several months to years after the onset of the wound.9,10 There are several pathophysiological factors that contribute to a wound entering into a chronic state, including:
• impaired angiogenic response;
• impaired macrophage function;
• impaired collagen formation in the wound bed; • increased pro-inflammatory cytokines at the wound bed disruption in the ratio of matrix metalloproteinases (MMPs) to tissue inhibitors of metalloproteinases (TIMPs); and • increased reactive oxygen species at the wound site.1,11
The gaseous signaling molecule hydrogen sulfide (H2S) plays a key role in many of these processes.
How Does Hydrogen Sulfide Act As A Signaling Molecule?
Hydrogen sulfide is a colorless gas with a characteristic rotten egg smell and an environmental pollutant that can inhibit cellular respiration at concentrations above 300 parts per million. In 1954, researchers discovered that various mammalian organ systems produced hydrogen sulfide.11-13 However, these original studies assumed that hydrogen sulfide was a metabolic waste product of no significance. This assumption prevailed until 1989 when scientists discovered hydrogen sulfide produced in mammalian brains, suggesting a possible physiological role for hydrogen sulfide in the body.15,16
We now know hydrogen sulfide is a key signaling messenger involved in several physiological processes in the body. All mammalian organ systems produce hydrogen sulfide through three key enzymes: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST).17-21
Much of the research looking at hydrogen sulfide’s role in regulating physiological processes has focused on the cardiovascular system. Hydrogen sulfide works in conjunction with nitric oxide to regulate vasodilation, reduce atherosclerotic plaque formation and decrease blood pressure.17,22,23 While hydrogen sulfide is known to be an important signaling molecule in the body, its role in the wound healing process has not been fully elucidated at this time.
How Might Hydrogen Sulfide Impact Multiple Wound Healing Processes?
Chronic diabetic wounds often exhibit impaired new blood vessel formation (angiogenesis).24-27 Vascular endothelial growth factor (VEGF) is responsible for stimulating endothelial cells to produce new blood vessels.28-30 In models of disease states with reduced levels of hydrogen sulfide, there is also diminished vascular endothelial growth factor-stimulated angiogenesis of endothelial cells.31,32 With restoration of hydrogen sulfide levels, however, there are significantly increased levels of vascular endothelial growth factor and angiogenesis by endothelial cells.33-36
One also finds reduced levels of hydrogen sulfide in models of obesity and diabetes.37-40 Hydrogen sulfide plays a key role in regulating angiogenesis, particularly in diabetic wounds. In a mouse model of diabetic wound healing, diabetic mice treated with hydrogen sulfide had greater numbers of capillaries in their wound beds in comparison to the control treatment, resulting in significantly faster rates of wound closure.41 Researchers attributed this to the activation of endothelial progenitor cells via increased expression of angiopoietin-1.41 In a diabetic rat model of wound healing, rats treated with a hydrogen sulfide donor showed significantly faster rates of wound closure in comparison to a vehicle control group.42 The rates of closure were similar to non-diabetic rats and the wound beds showed increased formation of granulation tissue and expression of vascular endothelial growth factor.42 Taken together, this indicates that hydrogen sulfide could improve diabetic wound healing by promoting angiogenesis at the wound site through upregulation of vascular endothelial growth factor.
One reason that diabetic foot ulcers fail to heal is that they are suspended in a state of chronic inflammation.1,9-11 This state of chronic inflammation leads to an increase in pro-inflammatory cytokines and oxidative stress at the wound, resulting in impaired macrophage function, disruption of the MMP/tissue inhibitors of metalloproteinases ratio and impaired collagen formation at the wound site.1,11 The gaseous signaling molecule hydrogen sulfide plays a key role in regulating inflammation and oxidative stress throughout the body.43-45
The anti-inflammatory effects of hydrogen sulfide may prove beneficial in stimulating chronic diabetic foot ulcers to heal. In a mouse model of wound healing, hydrogen sulfide treatment significantly reduced oxidative stress at the wound site, resulting in faster rates of wound closure in comparison to control groups.46 Furthermore, the group receiving exogenous hydrogen sulfide treatment had increased vascular endothelial growth factor expression at the wound site, resulting in greater capillary formation.46
Another study looking at diabetic wound healing in a mouse model found wounds from diabetic mice had increased levels of the pro-inflammatory cytokines IL-6 and TNF-α in comparison to non-diabetic controls.47 These mice also had increased recruitment of pro-inflammatory M1 macrophages at the wound site.47 Hydrogen sulfide treatment resulted in significantly reduced pro-inflammatory cytokine expression at the wound site as well as decreased macrophage recruitment. This resulted in faster rates of wound closure for the hydrogen sulfide-treated group in comparison to the control group. Through its actions as an antioxidant, hydrogen sulfide could promote wound healing by reducing oxidative stress at the wound site.
Macrophages are essential to the wound healing process by participating in the initial inflammatory response to debris and necrotic tissue in the wound bed. Macrophages initially take on a pro-inflammatory M1 phenotype to phagocytize cellular debris. As inflammation at the wound site resolves, macrophages transition to an anti-inflammatory M2 phenotype to help promote angiogenesis and establish a new extracellular matrix.48-50
In chronic wounds, however, the macrophages remain in a pro-inflammatory state and continue to degrade the extracellular matrix. In a mouse model of wound healing, mice treated with a hydrogen sulfide hydrogel showed decreased inflammation at the wound site, which resulted in a greater proportion of M2 macrophages in comparison to M1 macrophages.51 This resulted in greater collagen deposition and angiogenesis at the site, which ultimately led to faster rates of healing in hydrogen sulfide-treated group in comparison to the control group.51 Through its impact on macrophage polarization, hydrogen sulfide could transition chronic wounds into acute wounds, resulting in improved rates of wound healing.
What Is The Impact Of Hydrogen Sulfide On Matrix Metalloproteinases?
Matrix metalloproteinases (MMPs) play a vital role in the wound healing process. They are especially critical in the early stages of wound healing by removing damaged or degraded proteins, remodeling damaged portions of the extracellular matrix and promoting granulation tissue formation.52 In normal wound healing processes, MMP levels are carefully regulated. Throughout the wound healing process, MMP levels will decrease relative to tissue inhibitors of metalloproteinases.53,54 In chronic wounds, however, levels of MMPs remain elevated relative to levels of tissue inhibitors of metalloproteinases, resulting in a slow wound healing process that is likely due to continued degradation of the extracellular matrix.55,56
In chronic diabetic foot ulcers, high concentrations of MMP-9 correlate with increased bacterial load in the wound site and poor rates of wound healing.57 The antioxidant effects of hydrogen sulfide mitigate the expression of MMP-9 and restore the ratio of MMPs to tissue inhibitors of metalloproteinases. Microvessels isolated from obese animals had increased expression of MMP-9 relative to lean controls. When treated with a hydrogen sulfide donor, the expression of MMP-9 significantly attenuated to levels comparable to that of lean animals.40 Cystathionine γ-lyase is the primary enzyme responsible for producing hydrogen sulfide in the microvasculature. When vessels from lean animals were treated with a cystathionine γ-lyase enzyme inhibitor, the expression of MMP-9 significantly increased.40
In another study looking at the expression of MMP-9 in bone tissue homogenates, the authors found that treatment with hydrogen sulfide resulted in decreased MMP-9 expression.58 The authors proposed that the antioxidant role of hydrogen sulfide would decrease oxidative stress by reducing the levels of peroxynitrite, which is necessary for MMP activation. In their study, they proposed this would benefit bone healing as osteoclasts secrete MMP-9 to break down bone matrix.58 Another study looking at the effects of hydrogen sulfide on microvascular permeability in pial vessels in the brain found that hydrogen sulfide decreased microvascular permeability by reducing the expression and activity of MMP-9.59
Taken together, this data indicates that hydrogen sulfide could promote diabetic wound healing by reducing the activity and expression of MMP-9, thus restoring the MMP/tissue inhibitors of metalloproteinases ratio. Despite this fact, there is a lack of research exploring the impact of hydrogen sulfide on MMP activity, specifically in diabetic wound healing. It would be of interest to determine if hydrogen sulfide could promote wound healing by decreasing MMP activity and expression at the wound site.
Potential Pitfalls Of Hydrogen Sulfide In Wound Healing
One of the issues with using hydrogen sulfide as a wound healing modality is the limitation in controlling the release and rate of dissipation of hydrogen sulfide at the wound site. In a relatively recent study, Lin and colleagues looked at the use of hydrogen sulfide-containing microparticles to sustain a steady release of hydrogen sulfide at the wound site.60 The authors found that using a microparticle delivery system resulted in a slow and sustained release of hydrogen sulfide for up to 48 hours.60 The sustained release of hydrogen sulfide at the wound site resulted in improved angiogenesis at the wound site and faster rates of wound closure in comparison to controls in a mouse model of diabetic wound healing. This sustained delivery approach could have therapeutic potential for delivering hydrogen sulfide to wound sites.
Chronic diabetic foot ulcers remain a global health problem and a significant burden on health-care dollars. Failure to heal a diabetic foot ulcer in a prompt and timely manner can result in localized infection, sepsis and debilitating amputations for patients. Current treatment options, while beneficial, do not heal 100 percent of diabetic foot ulcers, resulting in serious consequences for patients as well as a burden on the health-care system. This warrants further research into other therapeutic targets that may be beneficial to the wound healing process.
One such target could be exploring the effects of gaseous signaling messengers such as hydrogen sulfide at the wound site. Several studies with animal models have shown that hydrogen sulfide could have a beneficial effect on the pathophysiology of diabetic wound healing. Hydrogen sulfide promotes angiogenesis at the wound site, reduces inflammation and oxidative stress, promotes polarization of macrophages to an anti-inflammatory phenotype and restores the ratio of MMPs to tissue inhibitors of metalloproteinases. Altogether, this indicates that hydrogen sulfide could promote closure of diabetic foot ulcers by stimulating wounds out of a chronic state, jump-starting the wound healing process.
To date, studies have not looked specifically at the role or safety of hydrogen sulfide on wound healing in human models. However, one study did examine the expression of the hydrogen sulfide-producing enzyme, cystathionine γ-lyase, in granulation tissues taken from diabetic and non-diabetic wounds in a human sample.37 The authors found that cystathionine γ-lyase enzyme expression was significantly lower in wounds from diabetic patients in comparison to non-diabetic patients.37 This indicates that in human diabetic foot wounds, there is decreased production of hydrogen sulfide. Perhaps by restoring hydrogen sulfide, we could promote wound closure. In conclusion, hydrogen sulfide may prove to be a useful and inexpensive tool in the diabetic wound healing arsenal.
Dr. Candela is a third-year resident with the Podiatric Residency Program with the Community Health Network in Indianapolis.
Dr. Baker is the Director of the Podiatric Residency Program with the Community Health Network in Indianapolis. He is in private practice in Anderson, Ind.
Dr. Koch is a faculty member of the Podiatric Residency Program with the Community Health Network in Indianapolis. She is in private practice in Anderson, Ind.
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