Of the six cytokines that showed a significant age-related difference, IL-6, IL-1β, IL-13, and VEGF are known to promote growth.
16–19 IL-10, while not directly linked to proliferation, is reported to act largely as an immune suppressor; however, other studies suggest it can also possess a proinflammatory function.
20 IL-10 expression has also been correlated with VEGF levels and it is proposed that IL-10 mediates VEGF expression,
21 which is known to stimulate growth in many cells,
18 and is also implicated in lens growth.
22 While there are differences between the age groups in the makeup of cytokines, one can still anticipate that the putative overall growth potential within the medium is similar in each age group. This is largely borne out by exposure of a human lens epithelial cell line, FHL124,
23,24 to conditioned medium pooled from young and older donors. However, the degree of stimulation observed was weak relative to the addition of 1% FCS. This, therefore, suggests that the contents of the medium are not the driving force of growth in the capsular bag. As a result, attention was drawn to the lens capsule and growth factors that associate with it. For our investigations, we selected growth factors that are established promoters of lens cell growth
25–27 and MAPK signaling.
25,28,29 For this purpose, we determined levels of bFGF
27,29 and HGF.
24,25 Moreover, bFGF and HGF require matrix association with heparan sulphate proteoglycans to facilitate receptor binding
30–34 and, interestingly, these become more prominent as the lens capsule thickens with increasing age.
35 It is possible that this elevated rate of protein synthesis observed in older capsular bags results from an overproduction of growth factors, etcetera, in an attempt to drive cell growth to rates approaching the young. We subsequently asked the question: Does downstream activity of signaling components account for age-related growth rates? To address this notion, we focused on several signaling pathways known to be mediated by growth factors that are commonly associated with growth and migration of lens and nonlenticular cells—namely p-ERK, p-p38, p-c-jun, and p-JNK.
36,37 Through application of pathway inhibitors to the human capsular bag system, it was apparent that coverage of the posterior capsule can be regulated by these pathways and thus demonstrates a common regulatory role. The overall reduction in signaling activity of ERK, p38, c-jun, and JNK correlates well with age-related differences in vitro and in the clinic and thus provides a rationale to explain these phenomena. As near optimal cell growth is observed in nonstimulated <40 years capsular bag cultures, we hypothesized that the signaling motif observed in these cells was close to a threshold of activity required to grow at a maximum rate. We therefore added serum as a global stimulation to promote multiple receptor responses in order to determine whether signaling activity could be enhanced in cells of aged capsular bags. In all cases, signaling activity was enhanced and thus explains the basis for serum rejuvenated growth in aged lens capsular bags. One explanation for the differences in signaling activity in serum-free medium is the reduced level of receptors in the cells of older donors relative to the young. However, when provided with sufficient ligand, in the form of serum, the rate of growth observed by the cells of aged capsular bags was driven to rates observed in young donors.
6 Moreover, MAPK signaling can be dramatically increased in response to serum of young and aged donors. This, therefore, suggests that receptor availability is not the limiting factor.