We found greater expression of hypoxia-related genes and smaller sizes of VAT than SAT adipocytes in obese subjects. The increased hypoxia in VAT is not likely to be a consequence of capillary rarefaction since vascular density, as well as EC number, were higher in VAT than SAT. However, the VAT-EC phenotype in obese subjects was markedly proangiogenic and inflammatory, with decreased expression of metabolism-related genes, including EL, GPIHBP1, and PPARγ. This phenotype of VAT-EC in obese subjects could be related to premature EC senescence, as suggested by expression of the senescence markers, IGFBP3 and γ-H2AX, as well as decreased expression of SIRT1.
AT is regionally distinct in terms of function, adipokine production, and inflammation. Adipocytes from VAT appear to have reduced capacity for lipogenesis (17
) and greater capacity for lipolysis than SAT cells (15
), with VAT containing more proinflammatory immune cells than SAT (12
). In the present study and consistent with other reports (18
), marked hypertrophy of SAT compared with VAT adipocytes was observed in obese subjects. Increased expression of the hypoxia-related genes, HIF-1α, VEGFA, and GLUT1 was found in VAT compared with SAT adipocytes. Despite previous studies reporting that leptin and FIAF are induced under hypoxic conditions (20
), we found they were lower in VAT than SAT. Irrespective of AT location, both leptin and FIAF transcript levels correlated with adipocyte size, as noted by others for leptin (22
). Expression of VEGFA and GLUT1 was tightly linked with that of HIF-1α. Moreover, SAT adipocytes maintained under low oxygen conditions had higher VEGFA and GLUT1 expresssion than VAT, whereas the expression of both leptin and FIAF was not affected substantially. Together, the present results indicate that hypoxia-related processes are more highly activated in VAT than SAT and are not related to the extent of adipocyte hypertrophy in obese subjects. Moreover, qualitative analyses by confocal microscopy and flow cytometry, using both the EC markers CD34 and CD31 simultaneously, reveal that capillary network density was higher in VAT than in SAT. Therefore, hypoxia in VAT is unlikely to be a consequence of capillary rarefaction.
Treatment of native SAT-EC originating from normal nonobese women with conditioned media originating from VAT adipocytes from obese subjects led to marked proliferation compared with conditioned media from SAT adipocytes from the same subjects. This suggests that the microenvironment of VAT is more proangiogenic than SAT and is consistent with the greater capillary density in VAT than SAT. Whether increased proangiogenic induction by VAT adipocytes is related to hypoxia- and inflammation-related events remains to be determined. Our results indicate that VAT-EC from obese subjects exhibited a markedly proinflammatory and angiogenic activated state, with increased expression of chemokines, adhesion molecules, and angiogenic factor receptors. This phenotype could contribute to the greater abundance of proinflammatory immune cells in VAT than SAT (12
). EL and GPIHBP1 are EC-specific metabolic genes, at least in murine models (23
). Together with PPARγ (the main regulator of GPIHBP1), these genes were downregulated in VAT compared with SAT. A recent study in murine models of diet-induced obesity highlighted the key role of PPARγ in the modulation of EC function and metabolic alterations associated with obesity (7
). Together our results show that although the capillary density of VAT is higher than that of SAT, VAT-EC exhibit a phenotype characterized by marked activation of inflammatory and angiogenic pathways associated with altered metabolic function in obese subjects.
This proangiogenic, proinflammatory phenotype might be related to premature endothelial cellular senescence. Indeed, senescent EC exhibit an activated state that may by induced either by extensive cell replication, leading to premature irreversible cell growth arrest, or by various stresses, including oxidative stress (24
). Decreased expression of SIRT1, together with increased expression of IGFBP3 and γ-H2AX, have been noted in senescent, replicatively exhausted human EC (25
), and we found indications of cellular senescence found in VAT-EC from obese subjects. Moreover, since conditioned media originating from VAT adipocytes increased the number of SA-β-gal positive EC, it is tempting to speculate that the chronic proangiogenic microenvironment of VAT promotes premature EC senescence, leading to endothelial dysfunction. Interestingly, VEGFA alone enhanced the number of SA-β-gal positive EC, although to a lesser extent than adipocyte-conditioned media. Moreover, neutralization of VEGFA reduced the senescence-promoting effect of the SAT and VAT adipocyte-conditioned media, suggesting that VEGFA might be an adipocyte-derived factor involved in premature adipose tissue EC senescence, consistent with its effects on EC senescence in other systems (29
Cellular senescence in other cell types is associated with a senescent secretory phenotype, with increased production of proinflammatory cytokines, chemokines, and extracellular matrix-modifying proteins, as well as angiogenic factors (31
). It will be important to define the secreted protein profile of senescent VAT-EC and SAT-EC and to determine whether such a phenotype is involved in the accumulation of immuno-inflammatory cells. Consistent with this possibility, we previously showed that treatment of SAT-EC with SAT adipocyte-conditioned media led to increased diapedesis of blood-derived monocytes (10
The subjects in our study were obese. Whether regional variation in fat tissue EC properties are already present in lean subjects or arise as a consequence of obesity needs to be determined. Since cellular senescence is increased in obesity (36
) and since obesity is associated with aortic endothelial cell senescence (37
), it is tempting to speculate that obesity could induce senescence in VAT-EC to a greater extent than in SAT-EC. Consistent with this speculation, our study shows that SAT-EC from lean and obese subjects did not exhibit marked phenotypic differences or numbers relative to AT weight. If comparative studies of VAT-EC from lean and obese subjects validate our speculation, interventions that limit the accumulation of senescent EC or their proinflammatory state could improve the approaches to limit inflammation attributable to obesity and its complications.