**3. Impaired wound healing observed in diabetes is associated with impaired microRNA expression**

### **3.1. Sustained inflammation: the role of mir-146a**

While inflammation plays an integral role in normal wound healing, the presence of pathologically sustained inflammation is a chief component of the dysregulated wound healing observed in patients with diabetes [15, 31]. The inflammatory phase of wound healing is characterized by increased infiltration of inflammatory cells (neutrophils and macrophages) and release of inflammatory mediators, such as cytokines [32]. Both the cellular and cytokine response to injury are mediated by microRNA. Specifically, miR-146a has been identified as a key regulator of the nuclear factor kappa-B (NFkB) pathway, which is known to regulate numerous inflammatory processes, as well as the transcription of several inflammatory cytokines [33, 34]. Following activation of the NFkB pathway by toll-like receptors (TLRs), NFkB expression is positively regulated by interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor 6 (TRAF6) [35]. Increased activity of IRAK1 and TRAF6 result in increased NFkB activity, which then upregulates the expression of genes coding for the key proinflammatory cytokines interleukin‐6 (IL‐6) and IL‐8 [17]. However, NFkB activity can also induce the expression of miR‐146a, which inhibits IRAK1 and TRAF6, thereby acting as a brake on the NFkB dependent innate immune response [36].

Analysis of skin samples obtained from wounded diabetic and nondiabetic mice demonstrates significant downregulation of the anti‐inflammatory miR‐146a in diabetic mice during the course of wound healing (**Figure 3**) [17]. In addition to down regulation of miR‐146a, wounded diabetic skin demonstrates significantly elevated expression of mRNA coding for IRAK1, TRAF6, NFkB, and the proinflammatory cytokines IL‐6 and IL‐8 (with MIP2 being the murine equivalent of IL‐8) (**Figure 3**) [17].

**Figure 3. Quantification of miRNA-146a and components of the NFkB pathway in diabetic and nondiabetic wounds.** Real‐time PCR quantification of miR‐146a **(A)**, IRAK1 **(B)**, TRAF6 **(C)**, NFkB **(D)**, and the downstream end products IL‐6 **(E)** and MIP2 **(F)** (the murine equivalent of IL‐8) days 0–21 after wounding in diabetic and nondiabetic mice. Results are presented as a mean + SEM for each cohort at each time point. Asterisk (\*) indicates *p* < 0.05 and # indicates *p* < 0.01. (Image reproduced from Xu *et al.* [17] with permission of the authors.)

### **3.2. Impaired biomechanical properties and deposition of extracellular matrix: the role of miR-29a**

In addition to dysregulation of the maturation phase of wound healing, diabetic skin has been shown to be biomechanically impaired at baseline, with decreased maximum load, maximum stress prior to failure, and decreased elasticity, as seen in **Figure 4** [21, 29]. It is thought this baseline impairment is one of the many factors that place even intact diabetic skin at a higher risk of injury than nondiabetic skin, with continued dysregulation of extracellular matrix remodeling contributing to impaired healing after injury [15, 29]. In addition, the balance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in diabetic wounds is weighted toward increased breakdown of extracellular matrix compo‐ nents, contributing to poor wound healing [2, 31, 37].

the key proinflammatory cytokines interleukin‐6 (IL‐6) and IL‐8 [17]. However, NFkB activity can also induce the expression of miR‐146a, which inhibits IRAK1 and TRAF6, thereby acting

Analysis of skin samples obtained from wounded diabetic and nondiabetic mice demonstrates significant downregulation of the anti‐inflammatory miR‐146a in diabetic mice during the course of wound healing (**Figure 3**) [17]. In addition to down regulation of miR‐146a, wounded diabetic skin demonstrates significantly elevated expression of mRNA coding for IRAK1, TRAF6, NFkB, and the proinflammatory cytokines IL‐6 and IL‐8 (with MIP2 being the murine

**Figure 3. Quantification of miRNA-146a and components of the NFkB pathway in diabetic and nondiabetic wounds.** Real‐time PCR quantification of miR‐146a **(A)**, IRAK1 **(B)**, TRAF6 **(C)**, NFkB **(D)**, and the downstream end products IL‐6 **(E)** and MIP2 **(F)** (the murine equivalent of IL‐8) days 0–21 after wounding in diabetic and nondiabetic mice. Results are presented as a mean + SEM for each cohort at each time point. Asterisk (\*) indicates *p* < 0.05 and #

**3.2. Impaired biomechanical properties and deposition of extracellular matrix: the role of**

In addition to dysregulation of the maturation phase of wound healing, diabetic skin has been shown to be biomechanically impaired at baseline, with decreased maximum load, maximum stress prior to failure, and decreased elasticity, as seen in **Figure 4** [21, 29]. It is thought this baseline impairment is one of the many factors that place even intact diabetic skin at a higher risk of injury than nondiabetic skin, with continued dysregulation of extracellular matrix remodeling contributing to impaired healing after injury [15, 29]. In addition, the balance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs)

indicates *p* < 0.01. (Image reproduced from Xu *et al.* [17] with permission of the authors.)

as a brake on the NFkB dependent innate immune response [36].

equivalent of IL‐8) (**Figure 3**) [17].

10 Wound Healing - New insights into Ancient Challenges

**miR-29a**

MiR‐29a contributes to this impaired extracellular matrix remodeling by posttranscriptional regulation of collagen content, leading to an inverse relationship between miR‐29a levels and collagen content [38, 39]. **Figure 5** details the significant upregulation of miR‐29a gene expression that has been detected in both diabetic murine (**Figure 5A**) and diabetic human skin (**Figure 5B**). This miR‐29a dysregulation corresponded to elevated gene expression of collagen 1α2 (col1α2) and collagen 3α1 (col3α1) in murine diabetic skin when compared with nondiabetic skin (**Figure 6**); however, Western blot confirmed decreased levels of both col1α2 and col3α1 protein in diabetic murine skin, versus nondiabetic murine skin (**Figure 6**). During the maturation phase of wound healing, the extracellular matrix undergoes remodeling

**Figure 4. Baseline biomechanical properties of diabetic and non-diabetic skin. (A)** The maximum load sustained **(N)** prior to failure in diabetic versus nondiabetic skin samples over 4–18 weeks of age. **(B)** The maximum stress to failure (MPa) in diabetic versus nondiabetic skin samples over 4–18 weeks of age. **(C)**: The elastic modulus (MPa) measured in diabetic versus non‐diabetic skin samples over 4–18 weeks of age. Data is presented as a mean + standard error of the mean (SEM) for each cohort. Student's *t*‐test was used to compare nondiabetic skin vs. diabetic skin at each time point. Asterisk (\*) indicates *p* < 0.05. Abbreviation: Max = Maximum. (Image reproduced from Zgheib *et al.* [21] with permis‐ sion of the authors.)

characterized by type III collagen being replaced by type I collagen, and this process is thought to be impaired in diabetic skin [15].

**Figure 5. MiRNA-29a gene expression in diabetic and nondiabetic murine (A)** and human **(B)** skin. **(A)** Real‐time quantitative PCR analysis of miRNA‐29a levels in murine diabetic and nondiabetic skin at different age‐points. **(B)** Real‐time quantitative PCR analysis of miRNA‐29a levels in human diabetic and nondiabetic skin. MiR‐29a gene ex‐ pression was calculated after normalizing with U6. Results are presented as a mean + SEM for each cohort. Student's *t*‐ test was used to compare nondiabetic skin to diabetic skin at each time point. Asterisk (\*) indicates *p* < 0.05. (Image reproduced from Zgheib *et al.* [21] with permission of the authors.)

The Role of MicroRNAs in Impaired Diabetic Wound Healing http://dx.doi.org/10.5772/63637 13

characterized by type III collagen being replaced by type I collagen, and this process is thought

**Figure 5. MiRNA-29a gene expression in diabetic and nondiabetic murine (A)** and human **(B)** skin. **(A)** Real‐time quantitative PCR analysis of miRNA‐29a levels in murine diabetic and nondiabetic skin at different age‐points. **(B)** Real‐time quantitative PCR analysis of miRNA‐29a levels in human diabetic and nondiabetic skin. MiR‐29a gene ex‐ pression was calculated after normalizing with U6. Results are presented as a mean + SEM for each cohort. Student's *t*‐ test was used to compare nondiabetic skin to diabetic skin at each time point. Asterisk (\*) indicates *p* < 0.05. (Image

reproduced from Zgheib *et al.* [21] with permission of the authors.)

to be impaired in diabetic skin [15].

12 Wound Healing - New insights into Ancient Challenges

**Figure 6. Collagen gene and protein expression in diabetic and non-diabetic murine skin. (A)** Relative gene expres‐ sion for collagen 1α2 in skin samples from diabetic (n = 5) and non‐diabetic (n = 5) mice from 4 to 18 weeks of age. **(B)** Relative gene expression for collagen 3α1 in skin samples from diabetic (n = 5) and non‐diabetic (n = 5) mice from 4 to 18 weeks of age. **(C)** Collagen I and III protein levels (upper band; black arrows) as demonstrated by western blots, obtained from skin samples from age‐matched, non‐diabetic and diabetic mice at 4 and 18 weeks of age. **(D)** Collagen I and III protein levels as quantified by western blot. These findings are representative of five independent experiments. Data is presented as a mean + SEM for each cohort. Student's *t*‐test was used to compare non‐diabetic skin to diabetic skin at each time point. Asterisk (\*) indicates p < 0.05; \*\* indicates p < 0.001. (Image reproduced from Zgheib *et al.* [21] with permission of the authors.)

### **3.3. Decreased angiogenesis: the role of miR-15b**

Successful angiogenesis requires coordinated extracellular matrix production in order to provide an adequate architecture for formation of new blood vessels [31]. However, angio‐ genesis within a wound bed is further regulated by numerous angiogenic factors, with vascular endothelial growth factor (VEGF) considered one of the most prominent [40]. Following injury, hypoxia in the wound bed leads to increased expression of hypoxia inducible factor‐1 (HIF‐1), a transcription factor that increases the expression of numerous proangiogenic proteins, including VEGF [31, 41]. In turn, VEGF attracts endothelial cells to the site of injury, in addition to inducing proliferation and angiogenesis via upregulation of proteins, such as the anti‐ apoptotic B‐cell lymphoma‐2 (BCL‐2) [16, 40]. BCL‐2 is also thought to improve wound healing by stabilizing the alpha subunit of HIF‐1 (HIF‐1α), mediated by heat shock protein 90 (HSP90), thereby increasing HIF‐1 mediated VEGF expression [16]. When compared to nondiabetic murine wounds, diabetic murine wounds have been detailed to have decreased levels of HIF‐1a activity, VEGF gene expression, and BCL‐2 gene expression, as well as significantly decreased number of cells that stain for the presence of platelet endothelial cell adhesion molecule 31 (CD31), a marker of endothelial cells [42].

MiR‐15b negatively regulates angiogenesis by decreasing VEGF expression; this decrease in VEGF expression is associated with decreased cell migration and vascular tubule formation

**Figure 7. Quantification of miRNA-15b, BCL2, and VEGFα gene expression in diabetic and non-diabetic wounds.** Quantification by real‐time PCR of miRNA‐15b (miR‐15b) **(A)**, BCL2 **(B)**, and VEGFα **(C)** gene expression in murine diabetic wounds versus murine non‐diabetic wounds, 3–7 days after wounding. Results are presented as a mean + SEM for each cohort at each time point. Asterisk (\*) indicates *p* < 0.05 and \*\* indicates *p* < 0.001. (Image repro‐ duced from Xu *et al.* [16] with permission of the authors.)

*in vitro* [43]. In nondiabetic humans and mice, the hypoxic conditions following wounding decrease the expression of miR‐15b, leading to increased levels of HIF‐1α, VEGF, and BCL‐2. However, in our murine model of diabetic wound healing, miR‐15b expression was signifi‐ cantly upregulated in diabetic mice compared to nondiabetic mice 1, 3, and 7 days after wounding (**Figure 7**). Furthermore, the upregulation in miR‐15b expression observed in diabetic mice was associated with a significant downregulation in VEGF and BCL‐2 gene expression 3 and 7 days after wounding (**Figure 7**).
