The ominous triad of adipose tissue dysfunction: inflammation, fibrosis, and impaired angiogenesis (original) (raw)
There is a physiologic limit to adipocyte cell size. Adipocytes in the mouse inguinal and epididymal fat pads can increase in size three- and seven-fold, respectively, during 12 weeks of HFF (62). Cell size correlates strongly with the frequency of adipocyte death. The percentage of dead epididymal adipocytes increases progressively from 0.1% at one week to as much as 16% at 12 weeks of HFF (62). Independent of how adipocytes die — from necrosis or apoptosis — the reaction of AT to adipocyte death can be likened to the initiation of a wound healing response, triggering a considerable increase in immune cell infiltration. Monocyte recruitment and differentiation to proinflammatory macrophages are of particular importance. These macrophages surround the dead adipocytes, forming what is described histologically as “crown-like structures” (62–64). Concomitantly, activated myofibroblasts in the area secrete collagen to maintain the integrity of the damaged tissue (65). As macrophages and neutrophils clean the damaged area, they produce toxic products such as ROS and reactive nitrogen species (RNS) that further injure surrounding cells and promote fibrosis. Dermal white AT plays an important role in topical wound healing, with the adipocytes as critical mediators (66). Inflammation during wound healing is self-limiting, provided the force producing the injury signal is removed. In fact, early removal of macrophages is essential to proper wound healing and prevention of scarring (67). However, in obesity the injury signal persists, causing chronic activation of myofibroblasts and immune cells, resulting in further tissue damage, fibrosis, and ultimately AT dysfunction.
Unresolved inflammation. The regulation of immune cell function and cytokine production in obese AT has been well studied (68), and it is now evident that the balance of pro- and antiinflammatory signals is critical for disease progression. Although a proinflammatory program is activated during early AT expansion, the immune response is dominated by antiinflammatory signals. During chronic obesity, both innate and adaptive immunity are activated and skewed to a proinflammatory response triggered by adipocyte death, hypoxia, and reduced fatty acid storage capacity in dysfunctional adipocytes.
One of the best studied consequences of proinflammatory signaling in AT is insulin resistance. TNF-α, produced by immune cells, was the first cytokine demonstrated to directly impede insulin action in the adipocyte (69). It downregulates the major insulin-responsive glucose transporter GLUT4 and inhibits insulin-dependent tyrosine phosphorylation of the insulin receptor and IRS-1 through ceramide production (70, 71). Other factors produced by inflammatory cells have since been shown to inhibit insulin signaling in the adipocyte including IL-6, IFN-γ and CCL2 (72, 73). Adaptive immune cells also produce factors that influence insulin signaling. B cells are divided into two main subfamilies, B1 and B2. B1 cells produce germline-encoded natural IgM and IgA antibodies. B2 cells are responsive to T cells upon antigen stimulation and produce a host of different adaptive antibodies as well as cytokines. In addition to cytokines, B2 cells also produce over a hundred different antibodies that have been associated with insulin resistance in human AT (8, 9).
It has been suggested that the production of ROS and RNS by immune cells contribute to the oxidative damage observed in obese AT (74, 75). Although this theory has yet to be directly tested, the degree of macrophage infiltration into obese AT and what we know about macrophage function in wound healing make this a plausible scenario. During the inflammatory phase of wound healing, activation of monocytes and macrophages induces a “respiratory burst,” in which large quantities of superoxide are generated by a membrane-bound NADPH oxidase. This respiratory burst precedes the production of multiple ROS including hydroxyl radicals and the longer-lived pro-oxidant hydrogen peroxide. This response makes macrophages lethal to pathogens, but it does not come without collateral damage. Macrophages need to be removed before surrounding tissues can be repaired (76). Increased oxidative stress and reduced antioxidant defense have been demonstrated in obese AT (74). Further work will be needed to determine whether this is a direct consequence of AT macrophage activation.
Interestingly, in humans, oxidative stress may precede AT inflammatory cell infiltration. Men fed a high-calorie diet for one week gained significant weight accompanied with insulin resistance (77). Oxidative damage was detected in AT biopsies without an increase in inflammatory cell markers. In mice, the chronology of oxidative stress and inflammation onset is not known; however, treatment of mice with antioxidants can attenuate diet-induced inflammation and insulin resistance, suggesting a strong influence of oxidative stress on obesity-associated disease (78–80). In addition, oxidative modification of cellular components such as glutathionylated 4-hydroxy-2-nonenal can directly stimulate a macrophage-mediated proinflammatory response (81).
Although the detrimental consequences of proinflammatory signaling in obese AT are evident, our group has shown that it is also required for appropriate expansion of AT and safe storage of potentially toxic lipid species (82). In fact, TNF-α plays an essential role in the adaptation of AT to HFF. Adipose-specific overexpression of a dominant-negative form of TNF-α causes severe glucose intolerance and a reduction in the insulin-sensitizing adipokine adiponectin in mice fed a HFD (82). Thus, AT inflammation in obese individuals cannot be assigned a strictly pathologic role.
Impaired angiogenesis. Under the persistent metabolic challenge of chronic HFF, the demand for oxygen is great but the capacity to form new blood vessels is poor. The consequence is disorganized and pathologic angiogenesis; however, the underlying mechanisms are not fully understood. Long-term HFF results in abnormal regulation of VEGF-A expression; not only do the VEGF-A levels decrease in the AT of obese mice and humans (13), but there is also evidence that the presence of VEGF in obese mice can block the regulation of neovascularization and vessel normalization (83). Interestingly, unlike acute AT expansion, the hypoxia response of obese ob/ob mice fails to induce VEGF expression, and instead a decrease in vascular density is observed (4). Although the majority of studies have shown that obese AT is hypoxic, we do not know if the degree of hypoxia is sufficient to enforce persistent angiogenesis, particularly in human AT. Indeed, it has been argued that the oxygen tension in AT is not low enough to be defined as hypoxic (84). Regardless of the intensity of the hypoxia, our studies have shown that activating the classic hypoxia response is not sufficient to ameliorate angiogenic deficiencies in obese mice. Genetic introduction of dominant-active HIF-1α in mice was unable to induce an angiogenic response but instead increased the expression of several fibrotic genes (4). Conversely, either overexpression of a dominant-negative HIF-1α or pharmacologic inhibition of HIF-1α significantly reduced AT fibrosis and improved AT function in mice fed a HFD (85). Further efforts are required to understand how AT angiogenesis is disrupted in obesity and how this can be therapeutically overcome.
One potential determinant of vessel density and integrity is the type of angiogenesis that occurs during long-term obesity. We can speculate that, as a result of myofibroblast activation in obese AT, new vascular networks are formed frequently through intussusception, or splitting of existing vessels. This rapid form of angiogenesis can be beneficial in the special conditions of cutaneous wound healing but may contribute to vascular dysfunction in obese fibrotic AT. Mechanical forces that exist during the rapid growth of a tumor induce this kind of angiogenesis (86). As in obese AT, tumor vasculature is significantly more permeable than that of normal tissue (87), a characteristic that promotes immune cell extravasation and fibrosis. Thus, future therapeutic strategies will benefit from an understanding of the mechanisms behind the pathologic angiogenesis observed in obesity.
Given the importance of appropriate vascularization for AT expansion, angiogenesis has been proposed as a potential therapeutic target to treat obesity and its related metabolic problems. Chemical angiogenic inhibitors such as angiostatin, endostatin, and thalidomide as well as the VEGFR2-blocking antibody TNP-470, have been tested in diet-induced obese mice or genetically obese ob/ob mice and shown to significantly reduce body weight and fat pad mass (88, 89). Additionally, a recent study suggested treatment with docosahexaenoic acid might improve insulin resistance through attenuation of AT angiogenesis (90). Several natural products extracted from plants (91–93) have also effectively reduced body weight in ob/ob and HFD-induced obese mice by inhibiting angiogenesis. Despite these results, the inhibition of angiogenesis as a therapeutic strategy for obesity remains a controversial notion (94). Any metabolic improvements afforded by these systemically introduced compounds cannot be directly attributed to reduced angiogenesis in AT. Our group has reported that WAT-specific overexpression of VEGF-A resulted in differential metabolic effects in mice challenged with a HFD or mice that are genetically obese (22). In HFD-challenged mice, VEGF-A significantly increased WAT vascularization and beiging, which augmented energy expenditure and prevented unfavorable metabolic changes, while blockade of VEGF-A/VEGFR2 caused aggravated systemic insulin insensitivity. In contrast, in ob/ob mice with pre-existing obesity, inhibiting the VEGF-A/VEGFR2 signaling pathway resulted in improved insulin sensitivity and decreased body weight.
Several key questions about the role of angiogenesis in AT remain. First, what types of pro-angiogenic mechanisms are in effect either simultaneously or sequentially (and in which order) during the expansion of AT? Second, how do we properly define the stages of AT expansion and further identify when angiogenesis is appropriate and when it is pathologic? Third, can modulators of angiogenesis be activated locally in different fat pads and achieve distinct effects? And finally, would an angiogenic strategy be more effective if combined with other existing anti-obesity therapies?
Fibrosis and ECM dysfunction. Fibrosis occurs during chronic obesity and has been accepted as a major contributor to obesity-associated AT dysfunction. Although deposition of fibrous collagen proteins is well known to promote metabolic dysfunction in obesity, many other ECM components are dysregulated in obese AT. Recent studies have shown an important role of microfibril-associated glycoprotein 1 (MAGP1) in obese AT. MAGP1 binds active TGF-β, sequestering it in the ECM (95). The actions of MAGP1 have been shown to protect mice from obesity and associated metabolic defects (95). Additionally, HA accumulates in insulin-resistant AT in obese mice and is thought to negatively regulate adipocyte insulin signaling (96). Fibrosis has been suggested to limit AT angiogenic capacity. Genetic knockout of the collagen-binding receptor integrin in the muscle of diet-induced obese mice increases vascularization (97).
Interestingly, the detrimental effects of fibrosis-mediated restriction of AT expansion during obesity on metabolic health may be dependent on the adipose depot affected. Mice genetically deficient in interferon regulatory factor 5 display enhanced expansion of subcutaneous AT but limited expansion of visceral AT on a HFD (98). Restricted epididymal AT expansion was associated with a large increase in antiinflammatory macrophage infiltration, collagen deposition, and improved insulin sensitivity. Thus, the ability of fibrosis to affect AT processes and signaling pathways in a depot-specific manner makes it a fertile topic for future research.
An integrated response: a role for cellular senescence in obesity. Recent studies have identified an important role for cellular senescence in many diseases including those associated with obesity. Senescent cells can originate from most cell types and are potent modulators of inflammation, angiogenesis, and fibrosis. Cells undergo so-called replicative senescence during natural aging when they have reached the genetically determined limit of division. Division-competent cells can also be pushed to senescence though damaging stresses such has oncogene induction, oxidative stress, and double-strand DNA breaks (99). Although growth arrested, senescent cells are highly metabolic and exhibit a senescence-associated secretory phenotype (SASP), producing factors that have profound effects on neighboring cells such as proinflammatory cytokines (IL-6, IL-8, TNF-α, monocyte chemoattractant protein 1 [MCP-1]), VEGF, MMPs, and PDGF-AA (100). Targeting senescent cells or their products alleviates age-related dysfunction of adipocyte progenitors and metabolism, as elegantly demonstrated in mice expressing a p16INK4a promoter–driven inducible caspase-8 (INK4-ATTAC mice) (101). In this model, the loss of senescent cells blunted age-related fat loss and enhanced adipogenic transcription factor expression within three weeks.
Little is known about the role of cellular senescence in obesity-associated disease. However, several studies suggest that senescence may be a contributing factor (Figure 2). Increased AT DNA damage and resulting cellular senescence have been identified in mice fed a HFD for 20 weeks (102). This study established a strong link between cell senescence and obesity by demonstrating that inducing genomic instability through ablation of polymerase η increased the number of senescent AT cells and exacerbated AT dysfunction. The prevention of cellular senescence through inhibition of the major senescence regulator p53 attenuates metabolic abnormalities (102, 103). Obesity is thought to promote cell senescence through various means including oxidative stress, high glucose concentrations in the microenvironment, and increased IGF and ceramides (104). Surprisingly, preadipocytes and mature adipocytes as well as endothelial cells can become senescent during obesity (105–109). Reduced blood flow, as seen in obese AT, is sufficient to trigger endothelial cell senescence (105, 110). Systemically, the circulating endothelial cell precursor population undergoes premature senescence in obese individuals (107), contributing to the impairments in vascular function and repair observed in obesity. Thus, cell senescence of adipocytes, endothelial cells, or their precursors might have major effects on AT homeostasis.
The role of senescence in obesity-associated AT dysfunction. Chronic obesity can cause AT oxidative stress, DNA damage, and increased exposure to high glucose and ceramide concentrations. These deleterious factors can drive cellular senescence in many cells types. Adipocyte and endothelial cell senescence have been specifically studied in the context of obesity. Mature adipocytes and endothelial cells as well as their precursors can undergo senescence and take on the SASP. SASP factors can promote AT dysfunction through dysregulation of AT ECM remodeling, inflammation, and angiogenesis.
SASP factors are also likely to have considerable effects on AT immunity, angiogenesis, and fibrosis. SASP has recently been shown to have a beneficial effect on wound healing by recruiting immune cells to clear dead cells, promote angiogenesis, limit fibrosis, and stimulate wound closure (100, 111, 112). In contrast, the proinflammatory cytokines released from senescent cells, including MCP-1, TNF-α, and IL-1β, are among those considered most important for the progression to dysfunctional AT and chronic fibrotic disease (65, 113). Therefore, in chronic obesity the beneficial effects of time-limited cellular senescence in wound healing are replaced by the detrimental effects of chronic SASP.
AT inflammation, fibrosis, and angiogenesis in human obesity. Although rodent models are crucial for our continued understanding of adipocyte biology in obesity, the translation of these findings to human disease is the ultimate goal. Briefly outlined below are findings from human studies on this subject.
Inflammation. Weisberg et al. demonstrated that subcutaneous AT macrophage infiltration was increased in obese patients, similar to that observed in obese mice (10). Several detailed studies have also reported a higher macrophage count in visceral AT of insulin-resistant obese patients compared with lean or insulin-sensitive obese counterparts, an effect that was significantly mitigated following bariatric surgery (10, 114–116). However, conflicting findings concerning AT inflammation in humans have also been reported. Boden et al. showed that healthy men can become insulin insensitive during acute excessive caloric intake but do not display inflammatory changes in AT (77). Moreover, others have shown that 12 months after bariatric surgery, AT inflammation remained elevated in approximately 40% of patients (117). Therefore, whether inflammation is the trigger or result of obesity-associated metabolic defects remains an important area for human studies.
Angiogenesis. Similar to findings in mice, human studies have reported that VEGF expression is reduced in obese humans (13). Consistent with mouse models of adipocyte-specific overexpression of VEGF-A, higher VEGF-A expression correlated with improved capillary density and insulin sensitivity in non-diabetic obese individuals (118). However, contradictory reports have demonstrated elevated levels of VEGF in obese subjects as well as a positive correlation between VEGF expression and insulin resistance (43). Thus, in both rodent models and human studies, VEGF-A–mediated AT angiogenesis exerts dichotomous effects in AT expansion and function.
Fibrosis. The majority of human studies have reported fibrosis in obese AT (119–121). However, a recent human study compared obese patients with or without T2D and found significantly less fibrosis in the visceral AT of the diabetic subjects (122). Another independent study also reported less collagen content in visceral AT from metabolically unhealthy obese patients (123). Less fibrosis was associated with adipocyte hypertrophy, reduced pre-adipocyte hyperplasia, and AT dysfunction (122). Thus, the role of ECM remodeling and fibrosis in human AT dysfunction is stage dependent and likely fat depot specific. This debate is also a reflection of the fact that no consensus has emerged as to how we should define fibrosis. Is it the total tissue collagen content? Is it a reflection of how intensely the collagen bundles are cross-linked? Is it about interstitial pericellular collagen accumulation, or is it about the density of the septa that separate individual functional units within adipose depots (124)?