Sexual dimorphic impact of adult-onset somatopause on life span and age-induced osteoarthritis

Abstract

Osteoarthritis (OA), the most prevalent joint disease, is a major cause of disability worldwide. Growth hormone (GH) has been suggested to play significant roles in maintaining articular chondrocyte function and ultimately articular cartilage (AC) homeostasis. In humans, the age-associated decline in GH levels was hypothesized to play a role in the etiology of OA. We studied the impact of adult-onset isolated GH deficiency (AOiGHD) on the life span and skeletal integrity including the AC, in 23- to 30-month-old male and female mice on C57/BL6 genetic background. Reductions in GH during adulthood were associated with extended life span and reductions in body temperature in female mice only. However, end-of-life pathology revealed high levels of lymphomas in both sexes, independent of GH status. Skeletal characterization revealed increases in OA severity in AOiGHD mice, evidenced by AC degradation in both femur and tibia, and significantly increased osteophyte formation in AOiGHD females. AOiGHD males showed significant increases in the thickness of the synovial lining cell layer that was associated with increased markers of inflammation (IL-6, iNOS). Furthermore, male AOiGHD showed significant increases in matrix metalloproteinase-13 (MMP-13), p16, and β-galactosidase immunoreactivity in the AC as compared to controls, indicating increased cell senescence. In conclusion, while the life span of AOiGHD females increased, their health span was compromised by high-grade lymphomas and the development of severe OA. In contrast, AOiGHD males, which did not show extended life span, showed an overall low grade of lymphomas but exhibited significantly decreased health span, evidenced by increased OA severity.

Keywords: articular cartilage; growth hormone; health span; life span; osteoarthritis; osteophyte; subchondral bone.

FIGURE 1

Effects of adult‐onset isolated growth hormone deficiency (AOiGHD) on NMR‐based body composition and life span. (a) AOiGHD model is based on the expression of a Cre‐recombinase under the rat GH promoter (rGHp), which releases a stop‐cassette upstream of a diphtheria toxin (DT) receptor (DTR) transgene in GH expressing somatotrophs in the pituitary. Upon DT injection, somatotrophs expressing DTR (rGHp‐Cre^+/−; DTR^+/−) are programmed for death, resulting in dramatic reductions in GH secretion and subsequently AOiGHD. (b) Male and (c) female mice were followed for body weight and body composition longitudinally. Body composition was examined monthly from 8 to 33 months by NMR. Presented are body weights, absolute and relative lean and fat mass, in male and female mice. (d) Life span was determined in male and female mice using Kaplan–Meier test. Sample size; male control n = 26, male AOiGHD n = 20, female control n = 25, female AOiGHD n = 17. Number of mice in each group dropped with age. (e) Kaplan–Meier data table showing life span, median survival and maximal life span of mice. (f) Rectal temperatures were monitored between 50 and 70 weeks of age in control male (n = 19–23), AOiGDH male (n = 16–19), control female (n = 10–15), and AOiGDH female mice (n = 13–17). Values are given as mean ± SEM; and *p < 0.05, **p < 0.01, and ***p < 0.001

FIGURE 2

End‐of‐life pathology. Pathology examination was done for 86 mice (control male (n = 25), AOiGDH male (n = 19), control female (n = 25), and AOiGDH female mice (n = 17)). (a) Percent of mice with lymphomas in the various tissues did not differ significantly between controls and AOiGHD mice. (b) Total number of tissues with lymphomas, (c) number of tissues with max grade lymphomas, or (d) number of tissues with average grade lymphomas were significantly higher in female than male mice but did not vary with genotype. When analyzed by tissue, control males (dark blue) showed increased (e) max grade and (f) average grade lymphomas (specifically in kidney, liver, spleen, pancreas, lung, adrenal glands and lymph nodes; Lns). Data are presented as mean ± SEM, *p < 0.05, **p < 0.01, and ***p < 0.001. M and F indicate male and female, respectively

FIGURE 3

AOiGHD males show significant degradation of the AC and increases in osteophyte formation. (a) Representative toluidine blue stained sections of the knee joint. AC: articular cartilage, L: ligament, MN: meniscus, and OS: osteophyte. (b) Cartilage loss was quantified by OARSI scoring system in femur and tibia (control male (n = 22), AOiGDH male (n = 15), control female (n = 19), and AOiGDH female mice (n = 13)). (c) micro‐CT 3D images of the knee joint, osteophytes are indicated by red arrows. (d) Osteophytes were quantified in femur and tibia from toluidine blue stained sections according to the OARSI scoring system (control male (n = 22), AOiGDH male (n = 15), control female (n = 20), and AOiGDH female mice (n = 14)). Data are presented as mean ± SEM; ^ns non‐significant, *p < 0.05, **p < 0.01, and ***p < 0.001. M and F indicate male and female, respectively

FIGURE 4

AOiGHD males show significant increases in synovitis (a) Representative H&E‐stained sections of the knee joint. Red arrows indicate the synovial membrane. (b) The thickness and cell density of the synovial cell lining layer were quantified by OARSI scoring system (control male (n = 21), AOiGDH male (n = 17), control female (n = 25), and AOiGDH female mice (n = 16)). (c) Representative images and quantification of the synovial membrane immunostained with IL6 and iNOS. MN; meniscus, SYN; synovium. (d) Synovial layer positive cells for IL6 and iNOS (control male (n = 7), AOiGDH male (n = 7), control female (n = 7), and AOiGDH female mice (n = 7)) Values given in mean ± SEM; ^ns non‐significant. M and F indicate male and female, respectively

FIGURE 5

AOiGHD does not affect the thickness or mineral density of the SCB plate. (a) The thickness and mineral density of the SCB plate of the tibia (LMTP) was quantified using mCT. (b) Representative 3D images of SCB at the distal femur and proximal tibia. (c) Quantification of SCB volume/total volume (BV/TV), bone mineral density (BMD), trabecular thickness (Tb. Th), and trabecular number (Tb.N) at the distal femur and (d) proximal tibia. (e) SCB parameters in the distal femur and proximal tibia as a function of age. Values given in mean ± SEM; ^ns non‐significant. M and F indicate male and female, respectively (control male (n = 21), AOiGDH male (n = 20), control female (n = 23), and AOiGDH female mice (n = 15)

FIGURE 6

AOiGHD male mice show increased inflammation and senescence in the AC. (a) MMP‐13, (b) NLRP3, (c) IL6, (d) iNOS, (e) p‐16, and (f) β‐galactosidase. Data presented as mean ± SEM; ^ns non‐significant, *p < 0.05, **p < 0.01, and ***p < 0.001. M and F indicate male and female, respectively (control male (n = 5–7), AOiGDH male (n = 5–7), control female (n = 5–7), and AOiGDH female mice (n = 5–7)). (g) Representative images of the articular cartilage immunostained with the aforementioned antibodies. Scale bar, 50 µm