**3. Skin aging**

#### **3.1 Mechanisms of skin aging**

Skin aging dependent on age or photoaging represents actually a real challenge of the new ADSCs advancements. The apparent skin physiology and morphology represent the first signal of cell aging. Skin aging is represented by epidermal atrophy, wrinkles appearance, reduction of dermal thickness, and ECM degradation; adnexal structures also decrease in their number and function. Decrease in the number of epithelial cells including DF, melanocytes, and Langerhans cells was reported as well. Their replicative ability decreases with age, leading to senescent and non-dividing cells.

The thickness of subcutaneous adipose tissue is also reduced, showing a decrease in mitotic activity and self-renewal of ADSCs and increase in senescence of the surrounding cells. ADSCs might behave differently according to the context of stimulation, but the mainly important factors that are relayed to cell proliferation, proinflammation, and angiogenesis altogether are involved in cell regeneration.

#### **3.2 Physiological markers of skin aging**

We believe now that aging is associated to many intrinsic signaling pathway related to epigenetic factors, some genetic predispositions, and extrinsic factors such as ultraviolet radiations, air and water pollution, resulting in the impairment of skin integrity and youth. One could consider that UV/infrared (IR) irradiations might lead to more cell damage than intrinsic factors. Indeed, following IR irradiation, skin cells present different DNA damages. Also, wrinkles, elasticity loss, pigmentation dysfunction, or hyperkeratosis are the most common symptoms reflecting the visual extent of aging as a result of the progressive atrophy of the

**45**

*Adipose-Derived Stem Cells (ADSCs) and Growth Differentiation Factor 11 (GDF11)…*

hypertrophy and inflammation has been largely reported [28, 51, 52].

upregulation of α-SMA, MMP-1, and MMP-2 expression [25].

probably to the involvement of MMP production [57, 63, 64].

while P63 expression was reduced [66, 67].

**4. Role of ADSCs in skin regeneration**

dermis. These manifestations rely on the impairment of cell senescence, a multifactorial event leading to skin integrity loss. Collagen production decreases and its degradation increases, leading to a quantitative and structural change in collagen

In humans, GDF11 is connected to age-related diseases and its serum level is associated with the physiology of aging [47, 48]. Its circulating level has been associated with aging in many human organs [23, 49, 50]. This factor was found to be able to antagonize aging specifically [29] and its remarkable action against myocardial

During skin aging, keratinocytes, DF and although endothelial cells secrete ubiquitous endopeptidases able to degrade the ECM proteins and called matrix metalloproteinase (MMP). Degradation of collagen fibers occurs with the major protease present in human skin MMP-1 that degrades type I and III collagen, followed by MMP-3 and MMP-9 [53]. The specific tissue inhibitors of metalloproteinases (TIMPs) regulate MMP processes where their increase in levels during aging was not concomitant with that of TIMPs leading to intrinsic collagen deficiency and accelerated skin aging. Moreover, Patel et al. have suggested that the ratio of MMP/TIMPs could be considered as a biomarker of wound healing and aging [54]. In photoaging, elastic fiber disorganization and degradation were observed, mainly due to activation of MMP-2, MMP-3, MMP-9, MMP-12, and MMP-13 [45, 55, 56]. UVA-treated fibroblasts presented a typical senescence phenotype including

Behind the increase in MMP levels, another component generated in skin cells is represented by reactive oxygen species (ROS). When activating the mitogenactivated protein kinase (MAPK) family, transcriptional factors such as activator protein 1 (AP-1) and nuclear factor-kB (NF-kB) induce upregulation of MMP in DF and keratinocytes [57–60]. In addition, when cells aged, dysfunctions of mitochondrial electron transport chains and a decrease in mitochondrial activity were reported leading to higher production of ROS [61, 62]. Accumulation of ROS induced oxidative damages of structural proteins and lipoproteins, thus favoring cell senescence. On the other hand, this senescence might also be induced by the decline in replicative capacity due to DNA damage, including DNA methylation, histone deacetylation, or to chromatine architecture change and to gene expression which compromise the intended cellular function. The increase in mitochondrial ROS generation was also correlated to the decrease in DF size and spreading observed during progressive ECM degradation and impaired DF attachment due

ROS also regulated different biological mechanisms including generation of inflammatory responses. Indeed, aging is associated to an increased secretion of proinflammatory proteins such as interleukin-6 (IL-6) and matrix metalloproteinase (MMP)-9, leading to immune changes. Prolonged release of ROS in skin might

Some transcription factors such as P63 (P53 related protein) and P16INK4a are reported as indicators of keratinocytes senescence. Indeed, expression of P16INK4a positive cells increased with chronological aging in human dermis and epidermis

During normal development, skin regeneration is performed by the resident ADSCs providing for cellular turnover during skin homeostasis and repair after injury [41]. The basal layer is the skin location where these active multipotent stem

amplify the inflammatory injury and promote chronic inflammation [65].

*DOI: http://dx.doi.org/10.5772/intechopen.91233*

fibers, which impact the dermis structure [44–46].

#### *Adipose-Derived Stem Cells (ADSCs) and Growth Differentiation Factor 11 (GDF11)… DOI: http://dx.doi.org/10.5772/intechopen.91233*

dermis. These manifestations rely on the impairment of cell senescence, a multifactorial event leading to skin integrity loss. Collagen production decreases and its degradation increases, leading to a quantitative and structural change in collagen fibers, which impact the dermis structure [44–46].

In humans, GDF11 is connected to age-related diseases and its serum level is associated with the physiology of aging [47, 48]. Its circulating level has been associated with aging in many human organs [23, 49, 50]. This factor was found to be able to antagonize aging specifically [29] and its remarkable action against myocardial hypertrophy and inflammation has been largely reported [28, 51, 52].

During skin aging, keratinocytes, DF and although endothelial cells secrete ubiquitous endopeptidases able to degrade the ECM proteins and called matrix metalloproteinase (MMP). Degradation of collagen fibers occurs with the major protease present in human skin MMP-1 that degrades type I and III collagen, followed by MMP-3 and MMP-9 [53]. The specific tissue inhibitors of metalloproteinases (TIMPs) regulate MMP processes where their increase in levels during aging was not concomitant with that of TIMPs leading to intrinsic collagen deficiency and accelerated skin aging. Moreover, Patel et al. have suggested that the ratio of MMP/TIMPs could be considered as a biomarker of wound healing and aging [54]. In photoaging, elastic fiber disorganization and degradation were observed, mainly due to activation of MMP-2, MMP-3, MMP-9, MMP-12, and MMP-13 [45, 55, 56]. UVA-treated fibroblasts presented a typical senescence phenotype including upregulation of α-SMA, MMP-1, and MMP-2 expression [25].

Behind the increase in MMP levels, another component generated in skin cells is represented by reactive oxygen species (ROS). When activating the mitogenactivated protein kinase (MAPK) family, transcriptional factors such as activator protein 1 (AP-1) and nuclear factor-kB (NF-kB) induce upregulation of MMP in DF and keratinocytes [57–60]. In addition, when cells aged, dysfunctions of mitochondrial electron transport chains and a decrease in mitochondrial activity were reported leading to higher production of ROS [61, 62]. Accumulation of ROS induced oxidative damages of structural proteins and lipoproteins, thus favoring cell senescence. On the other hand, this senescence might also be induced by the decline in replicative capacity due to DNA damage, including DNA methylation, histone deacetylation, or to chromatine architecture change and to gene expression which compromise the intended cellular function. The increase in mitochondrial ROS generation was also correlated to the decrease in DF size and spreading observed during progressive ECM degradation and impaired DF attachment due probably to the involvement of MMP production [57, 63, 64].

ROS also regulated different biological mechanisms including generation of inflammatory responses. Indeed, aging is associated to an increased secretion of proinflammatory proteins such as interleukin-6 (IL-6) and matrix metalloproteinase (MMP)-9, leading to immune changes. Prolonged release of ROS in skin might amplify the inflammatory injury and promote chronic inflammation [65].

Some transcription factors such as P63 (P53 related protein) and P16INK4a are reported as indicators of keratinocytes senescence. Indeed, expression of P16INK4a positive cells increased with chronological aging in human dermis and epidermis while P63 expression was reduced [66, 67].

### **4. Role of ADSCs in skin regeneration**

During normal development, skin regeneration is performed by the resident ADSCs providing for cellular turnover during skin homeostasis and repair after injury [41]. The basal layer is the skin location where these active multipotent stem

*Regenerative Medicine*

induce skin rejuvenation [41–43].

**3.1 Mechanisms of skin aging**

and non-dividing cells.

**3.2 Physiological markers of skin aging**

**3. Skin aging**

substance. Both these components are rearranged to provide a three-dimensional microenvironment where epithelial cells, stem cells, and the vascular network are closely related to collagen, elastin, and fibronectin fibers [37]. In human skin, collagen fibers, mostly type I, III, and V, are the dominant components in the ECM accounting for 75% of the dry skin weight and confer elasticity and strength. Type I collagen represents 80–90% of the total collagen and type III up to 8–12% while type V collagen represents the remaining minor proportion [38]. ECM not only provides a structural support for skin cells but also plays very critical role in regulating cell behavior in normal conditions and wound healing [39]. This regulation occurs through molecular signaling mediated by integrin cell surface, which orients

cells toward proliferation, differentiation, migration, or apoptosis [40].

Additional skin components residing in the dermis and sometimes in the hypodermis are immune cells represented by lymphocytes, macrophages, mast and dendritic cells. Adnexal structures are located in the dermis and hypodermis and include hair follicles, blood vessels, nerves, eccrine glands, sebaceous glands, and apocrine gland. The hypodermis layer or subcutaneous layer is composed mostly by adipose tissue containing adipocytes, stem cells (ADSCs), and blood and lymph vessels. This adipose tissue is the main actor in regulating skin homeostasis, thermoregulation, metabolism, immune responses, and immunomodulation through a wide range of cytokines and chemokines secreted. This secretory panel conditions the microenvironment of the surrounding ADSCs and consequently their secretome to modulate skin cell proliferation, differentiation, migration, melanin production, and to

Skin aging dependent on age or photoaging represents actually a real challenge of the new ADSCs advancements. The apparent skin physiology and morphology represent the first signal of cell aging. Skin aging is represented by epidermal atrophy, wrinkles appearance, reduction of dermal thickness, and ECM degradation; adnexal structures also decrease in their number and function. Decrease in the number of epithelial cells including DF, melanocytes, and Langerhans cells was reported as well. Their replicative ability decreases with age, leading to senescent

The thickness of subcutaneous adipose tissue is also reduced, showing a decrease

in mitotic activity and self-renewal of ADSCs and increase in senescence of the surrounding cells. ADSCs might behave differently according to the context of stimulation, but the mainly important factors that are relayed to cell proliferation, proinflammation, and angiogenesis altogether are involved in cell regeneration.

We believe now that aging is associated to many intrinsic signaling pathway related to epigenetic factors, some genetic predispositions, and extrinsic factors such as ultraviolet radiations, air and water pollution, resulting in the impairment of skin integrity and youth. One could consider that UV/infrared (IR) irradiations might lead to more cell damage than intrinsic factors. Indeed, following IR irradiation, skin cells present different DNA damages. Also, wrinkles, elasticity loss, pigmentation dysfunction, or hyperkeratosis are the most common symptoms reflecting the visual extent of aging as a result of the progressive atrophy of the

**44**

cells are responsible for recruiting and sending mature differentiated cells (keratinocytes) to the outer layer of epidermis. Through a hierarchic gradient, these stem cells induced the regeneration of epidermis layer by ensuring self-renewal and a continuous production of transient amplifying cells [68]. Epidermal cells including ADSCs and DF closely interact to maintain local microenvironment propitious for cell turnover, leading to skin regeneration. Adding to their tendency to differentiate into keratinocytes, DF, and probably melanocytes, cross-talk of ADSCs and these cells is a part of normal skin function where ECM secretion leads to a physical environment critical for the maintenance of the stem cell niche [69].

Another proof of the interactions between fibroblasts and ADSCs has been provided by Hu et al. where skin fibroblasts cell line HS27 was found to activate ADSCs to differentiate into fibroblast-like cells highly expressing vimentin, HSP47, and desmin mRNA level [70]. The interactions of microvascular endothelial cells and ADSCs are also of great interest in skin cell regeneration and proliferation by providing IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and VEGF, leading to inflammation and angiogenesis regulation [71, 72]. In normal conditions, ADSCs are continuously activated by human serum and platelets to induce their proliferation and differentiation. While in wounded tissues, platelets induced stem cells to initiate the inflammatory phase by secreting platelet-derived growth factor (PDGF), IL-6 and IL-8, which lead to migration of macrophages and neutrophils to the wounded site [73], and TGF-β inducing induction of monocytes to macrophages (**Figure 1**).

#### **Figure 1.**

*Implication of adipose-derived stem cells in the different phases associated to wound healing and in the rejuvenation process. ADSCs act on fibroblasts, macrophages, and skin cells through their secreted growth factors. GDF11 and TGF-β are present in all the phases, amplify fibroblast, macrophage, and ADSC secretion, leading to immune response, cell proliferation, and angiogenesis. However, their interactions are more relevant during proliferation phases where GDF11 might induce TFG-β induction in a spatio-temporal manner in addition to boosting fibroblast proliferation, resulting in the production of skin cell presenting young profile. GDF11: growth differentiation factor, TGF-β: transforming growth factor, ECM: extracellular matrix, PDGF: platelets-derived growth factor, Il-1,6,8,10: interleukin-1, TNF-α: tumor necrosis factor-α, b-FGF: basicfibroblast growth factor, VEGF: vascular endothelial growth factor, CXCR-4: C motif chemokine receptor 4, SDF-1: stromal derived factor-1, TLR2, 4: toll-like receptor2,4, GM-CSF: granulocyte monocyte-colony stimulating factor, IGF: insulin growth factor, MMP-1,-2, -9: matrix metalloproteinase-1, -2, -9, α-SMA: α-smooth muscle actin.*

**47**

*Adipose-Derived Stem Cells (ADSCs) and Growth Differentiation Factor 11 (GDF11)…*

tory cytokine secretion by polarizing macrophages from M1 to M2.

Similar to platelets, ADSCs additionally secrete prostaglandin E2 (PGE2), TNF-α, and GDF11, thus potentiating proinflammatory responses and later anti-inflamma-

In addition to TGF-β and GDF11, ADSCs secrete other growth factors such as basic-fibroblast Growth Factor (b-FGF), stromal-derived factor-1 (SDF-1), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and wingless 10b (Wnt10b), which are involved in the mechanisms regulating skin cell regeneration and repair [10, 23, 74]. The factors VEGF, PDGF, TGF-β, b-FGF, and HGF induce the formation of new blood vessels during the proliferative phase, probably through their differentiation into endothelial progenitor cells [10, 75]. The secreted GM-CSF can take part in this activation by inducing differentiation into committed monocytes potentially activated to macrophages and endothelial cells. In chronic radiation wounds, the use of ADSCs promoted new blood vessels damaged by the irradiation and increased the capillary density. These cells were found to act through stimulation of fibroblast proliferation and increase in VEGF secretion

However, endothelial cells and macrophages migration have been possible once after macrophages leaded to secretion of the ECM proteins especially collagen I and III, elastin and fibronectin and to activation of DF to proliferate and migrate. DF also secretes VEGF, TGF-β, and b-FGF, leading to angiogenesis [75, 77]. ADSCs are expected to participate actively in ECM production, whereby its abundant accumulation would facilitate cell migration and angiogenesis by autoinduction and amplification of the growth factor secretion implied. When miming injured conditions *in vitro*, ADSCs were indeed demonstrated to accelerate neovascularization through the expression of hypoxia-inducible factor-1α [78] by regulating VEGF gene expression in endothelial cells [79]. These observations were confirmed *in vitro* in addition to an activation of stem cell proliferation and to keratinocytes chemoattraction and migration [80] likely facilitated by

Migration of ADSCs to the injured site is of pivotal interest; their immune profile and their potential shift toward a more anti-inflammatory phenotype is required for the proliferation and remodeling phases of healing [82–84]. The cytokine profile of T, B, and dendritic cells was influenced by ADSCs, which lead to the interruption of the inflammatory phase and starting the proliferation and remodeling phases in chronic wounds [6, 85, 86]. The potential involvement of GDF11 in disrupting the proinflammatory status toward the anti-inflammatory one is likely due to its highly stimulation of DF and ADSCs and also by amplifying the action of TGF-β on ADSCs proliferation and secretome. This epigenetic modification and regulation of ADSCs' microenvironment might be crucial in restoring cellular age defects and/or increasing cells' ability to differentiate and migrate. The impact of changing microenvironment on induction of cell proliferation, differentiation, and

The collagen production amplified by cross-talk between ADSCs, DF, and TGF-β would facilitate the remodeling phase through inhibiting ECM degradation by increasing TIMPs' secretion and their binding to MMPs [87]. Xiao et al. have reported that adipose tissue secretome increased N-cadherin and CD44 adhesion molecules involved in fibroblasts' motility during wound healing and stimulation of fibronectin expression during ECM remodeling [88]. Combination of activin B and ADSCs led to rapid wound closure and to accelerated epithelialization through promoting keratinocytes and fibroblasts proliferation [5]. The integrin αβ6 exclusively expressed by epithelial cells was associated to the regeneration of basement

**Figure 1** summarizes ADSCs' mechanisms involved in tissue repair.

*DOI: http://dx.doi.org/10.5772/intechopen.91233*

level [76].

MMPs [81].

migration has already been reported [41–43].

membrane zone during wound repair [89].

#### *Adipose-Derived Stem Cells (ADSCs) and Growth Differentiation Factor 11 (GDF11)… DOI: http://dx.doi.org/10.5772/intechopen.91233*

Similar to platelets, ADSCs additionally secrete prostaglandin E2 (PGE2), TNF-α, and GDF11, thus potentiating proinflammatory responses and later anti-inflammatory cytokine secretion by polarizing macrophages from M1 to M2.

In addition to TGF-β and GDF11, ADSCs secrete other growth factors such as basic-fibroblast Growth Factor (b-FGF), stromal-derived factor-1 (SDF-1), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and wingless 10b (Wnt10b), which are involved in the mechanisms regulating skin cell regeneration and repair [10, 23, 74]. The factors VEGF, PDGF, TGF-β, b-FGF, and HGF induce the formation of new blood vessels during the proliferative phase, probably through their differentiation into endothelial progenitor cells [10, 75]. The secreted GM-CSF can take part in this activation by inducing differentiation into committed monocytes potentially activated to macrophages and endothelial cells. In chronic radiation wounds, the use of ADSCs promoted new blood vessels damaged by the irradiation and increased the capillary density. These cells were found to act through stimulation of fibroblast proliferation and increase in VEGF secretion level [76].

However, endothelial cells and macrophages migration have been possible once after macrophages leaded to secretion of the ECM proteins especially collagen I and III, elastin and fibronectin and to activation of DF to proliferate and migrate. DF also secretes VEGF, TGF-β, and b-FGF, leading to angiogenesis [75, 77]. ADSCs are expected to participate actively in ECM production, whereby its abundant accumulation would facilitate cell migration and angiogenesis by autoinduction and amplification of the growth factor secretion implied. When miming injured conditions *in vitro*, ADSCs were indeed demonstrated to accelerate neovascularization through the expression of hypoxia-inducible factor-1α [78] by regulating VEGF gene expression in endothelial cells [79]. These observations were confirmed *in vitro* in addition to an activation of stem cell proliferation and to keratinocytes chemoattraction and migration [80] likely facilitated by MMPs [81].

Migration of ADSCs to the injured site is of pivotal interest; their immune profile and their potential shift toward a more anti-inflammatory phenotype is required for the proliferation and remodeling phases of healing [82–84]. The cytokine profile of T, B, and dendritic cells was influenced by ADSCs, which lead to the interruption of the inflammatory phase and starting the proliferation and remodeling phases in chronic wounds [6, 85, 86]. The potential involvement of GDF11 in disrupting the proinflammatory status toward the anti-inflammatory one is likely due to its highly stimulation of DF and ADSCs and also by amplifying the action of TGF-β on ADSCs proliferation and secretome. This epigenetic modification and regulation of ADSCs' microenvironment might be crucial in restoring cellular age defects and/or increasing cells' ability to differentiate and migrate. The impact of changing microenvironment on induction of cell proliferation, differentiation, and migration has already been reported [41–43].

The collagen production amplified by cross-talk between ADSCs, DF, and TGF-β would facilitate the remodeling phase through inhibiting ECM degradation by increasing TIMPs' secretion and their binding to MMPs [87]. Xiao et al. have reported that adipose tissue secretome increased N-cadherin and CD44 adhesion molecules involved in fibroblasts' motility during wound healing and stimulation of fibronectin expression during ECM remodeling [88]. Combination of activin B and ADSCs led to rapid wound closure and to accelerated epithelialization through promoting keratinocytes and fibroblasts proliferation [5]. The integrin αβ6 exclusively expressed by epithelial cells was associated to the regeneration of basement membrane zone during wound repair [89].

**Figure 1** summarizes ADSCs' mechanisms involved in tissue repair.

*Regenerative Medicine*

cells are responsible for recruiting and sending mature differentiated cells (keratinocytes) to the outer layer of epidermis. Through a hierarchic gradient, these stem cells induced the regeneration of epidermis layer by ensuring self-renewal and a continuous production of transient amplifying cells [68]. Epidermal cells including ADSCs and DF closely interact to maintain local microenvironment propitious for cell turnover, leading to skin regeneration. Adding to their tendency to differentiate into keratinocytes, DF, and probably melanocytes, cross-talk of ADSCs and these cells is a part of normal skin function where ECM secretion leads to a physical

Another proof of the interactions between fibroblasts and ADSCs has been provided by Hu et al. where skin fibroblasts cell line HS27 was found to activate ADSCs to differentiate into fibroblast-like cells highly expressing vimentin, HSP47, and desmin mRNA level [70]. The interactions of microvascular endothelial cells and ADSCs are also of great interest in skin cell regeneration and proliferation by providing IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and VEGF, leading to inflammation and angiogenesis regulation [71, 72]. In normal conditions, ADSCs are continuously activated by human serum and platelets to induce their proliferation and differentiation. While in wounded tissues, platelets induced stem cells to initiate the inflammatory phase by secreting platelet-derived growth factor (PDGF), IL-6 and IL-8, which lead to migration of macrophages and neutrophils to the wounded site [73], and TGF-β inducing induction of monocytes to macrophages (**Figure 1**).

*Implication of adipose-derived stem cells in the different phases associated to wound healing and in the rejuvenation process. ADSCs act on fibroblasts, macrophages, and skin cells through their secreted growth factors. GDF11 and TGF-β are present in all the phases, amplify fibroblast, macrophage, and ADSC secretion, leading to immune response, cell proliferation, and angiogenesis. However, their interactions are more relevant during proliferation phases where GDF11 might induce TFG-β induction in a spatio-temporal manner in addition to boosting fibroblast proliferation, resulting in the production of skin cell presenting young profile. GDF11: growth differentiation factor, TGF-β: transforming growth factor, ECM: extracellular matrix, PDGF: platelets-derived growth factor, Il-1,6,8,10: interleukin-1, TNF-α: tumor necrosis factor-α, b-FGF: basicfibroblast growth factor, VEGF: vascular endothelial growth factor, CXCR-4: C motif chemokine receptor 4, SDF-1: stromal derived factor-1, TLR2, 4: toll-like receptor2,4, GM-CSF: granulocyte monocyte-colony stimulating factor, IGF: insulin growth factor, MMP-1,-2, -9: matrix metalloproteinase-1, -2, -9, α-SMA:* 

environment critical for the maintenance of the stem cell niche [69].

**46**

*α-smooth muscle actin.*

**Figure 1.**
